Humanized antibodies to human gp39, compositions containing thereof

ABSTRACT

The present invention is directed to humanized antibodies which bind human gp39 and their use as therapeutic agents. These humanized antibodies are especially useful for treatment of autoimmune diseases.

FIELD OF THE INVENTION

The present invention is directed to humanized antibodies specific forhuman gp39, DNA encoding such antibodies, methods for their production,pharmaceutical compositions containing, and the use of such humanizedantibodies as therapeutic agents. These antibodies have particularapplication in the treatment of autoimmune diseases including, e.g.,rheumatoid arthritis, multiple sclerosis, diabetes, and systemic lupuserythematosus as well as non-autoimmune diseases including, e.g.,graft-versus-host disease and for preventing graft rejection.

BACKGROUND OF THE INVENTION

The immune system is capable of producing two types of antigen-specificresponses to foreign antigens. Cell-mediated immunity is the term usedto refer to effector functions of the immune system mediated by Tlymphocytes. Humoral immunity is the term used to refer to production ofantigen-specific antibodies by B lymphocytes. It has long beenappreciated that the development of humoral immunity against mostantigens requires not only antibody-producing B lymphocytes but also theinvolvement of helper T (hereinafter Th) lymphocytes. (Mitchison, Eur.J. Immunol., 1:18-25 (1971); Claman and Chaperon, Transplant Rev.,1:92-119 (1969); Katz et al., Proc. Natl. Acad. Sci. USA, 70:2624-2629(1973); Raff et al., Nature, 226:1257-1260 (1970)). Certain signals, or"help", are provided by Th cells in response to stimulation byThymus-dependent (hereinafter TD) antigens. While some B lymphocyte helpis mediated by soluble molecules released by Th cells (for instancelymphokines such as IL-4 and IL-5), activation of B cells also requiresa contact-dependent interaction between B cells and Th cells. (Hirohataet al., J. Immunol., 140:3736-3744 (1988); Bartlett et al., J. Immunol.,143:1745-1765 (1989)). This indicates that B cell activation involves anobligatory interaction between cell surface molecules on B cells and Thcells. Such an interaction is further supported by the observation thatisolated plasma membranes of activated T cells can provide helperfunctions necessary for B cell activation. (Brian, Proc. Natl. Acad.Sci. USA, 85:564-568 (1988); Hodgkin et al., J. Immunol., 145:2025-2034(1990); Noelle et al., J. Immunol., 146:1118-1124 (1991)).

It is further known that in a contact-dependent process termed "T cellhelper function", CD4⁺ T lymphocytes direct the activation anddifferentiation of B lymphocytes and thereby regulate the humoral immuneresponse by modulating the specificity, secretion and isotype-encodedfunctions of antibody molecules (Mitchell et al., J. Exp. Med., 128:821(1968); Mitchison, Eur. J. Immunol., 1:68 (1971); White et al., J. Exp.Med., 14:664 (1978); Reinherz et al., Proc. Natl. Acad. Sci. USA,74:4061 (1979); Janeway et al., Immunol. Rev., 101:39 (1988); O'Brien etal. J. Immunol., 141:3335 (1988); Rahemtulla et al., Nature, 353:180(1991); and Grusby et al., Science, 253:1417 (1991)).

The process by which T cells help B cells to differentiate has beendivided into two distinct phases; the inductive and effector phases(Vitetta et al., Adv. Immunol., 45:1 (1989); Noelle et al., Immunol.Today, 11:361 (1990)). In the inductive phase, resting T cells contactantigen-primed B cells and this association allows clonotypic T cellreceptor (TCR)-CD4 complexes to interact with Ia/Ag complexes on B cells(Janeway et al., Immunol. Rev., 101:39 (1988); Katz et al., Proc. Natl.Acad. Sci., 70:2624 (1973); Zinkernagel, Adv. Exp. Med., 66:527 (1976);Sprent, J. Exp. Med., 147:1159 (1978); Sprent, Immunol. Rev., 42:158(1978); Jones et al., Nature, 292:547 (1981);

Julius et al., Eur. J. Immunol., 18:375 (1982); Chestnut et al., J.Immunol., 126:1575 (1981); and Rogozinski et al., J. Immunol., 126:735(1984)). TCR/CD4 recognition of Ia/Ag results in the formation of stableT-B cognate pairs and bi-directional T and B cell activation (Sanders etal., J. Immunol., 137:2395 (1986); Snow et al., J. Immunol., 130:614(1983); Krusemeier et al., J. Immunol., 140:367 (1988); Noelle et al.,J. Immunol., 143:1807 (1989); Bartlett et al., J. Immunol., 143:1745(1989); and Kupfer et al., Annu. Rev. Immunol., 7:309 (1987)). In theeffector phase, activated T cells drive B cell differentiation bysecreting lymphokines (Thompson et al., J. Immunol., 134:369 (1985)) andby contact-dependent stimuli (Noelle et al., J. Immunol., 143:1807(1989); Clement et al., J. Immunol., 140:3736 (1984); Crow et al., J.Exp. Med., 164:1760 (1986); Brian, Proc. Natl. Acad. Sci., USA, 85:564(1988); Hirohata et al., J. Immunol. 140:3736 (1988); Jover et al.,Clin. Immunol. Immun., 53:90 (1989); Whalen et al., J. Immunol.,141:2230 (1988); Pollok et al., J. Immunol., 146:1633 (1991); andBartlett et al., J. Immunol., 143:1745 (1990)), both of which arerequired for T cells to drive small resting B cells to terminallydifferentiate into Ig secreting cells (Clement et al., J. Immunol.,132:740 (1984); Martinez et al., Nature, 290:60 (1981); and Andersson etal., Proc. Natl. Acad. Sci., USA, 77:1612 (1980)).

Although the inductive phase of T cell help is Ag-dependent andMHC-restricted (Janeway et al., Immun. Rev., 101:34 (1988); Katz et al.,Proc. Natl. Acad. Sci., USA, 10:2624 (1973); Zinkernagle, Adv. Exp. Med.Biol., 66:527 (1976)); the effector phase of T cell helper function canbe Ag-independent and MHC-nonrestricted (Clement et al., J. Immunol.,132:740 (1984); Hirohata et al., J. Immunol., 140:3736 (1988); Whalen etal., J. Immunol., 143:1715 (1988)). An additional contrasting feature isthat the inductive phase of T cell help often requires CD4 molecules andis inhibited by anti-CD4 mAb (Rogozinski et al., J. Immunol., 126:735(1984)), whereas helper effector function does not require CD4 molecules(Friedman et al., Cell Immunol., 103:105 (1986)) and is not inhibited byanti-CD4 mAbs (Brian, Proc. Natl. Acad. Sci., USA, 85:564 (1988);Hirohata et al., J. Immunol., 140:3736 (1988); Whalen et al., J.Immunol., 143:1745 (1988); and Tohma et al., J. Immunol., 146:2547(1991)). The non-specific helper effector function is believed to befocused on specific B cell targets by the localized nature of the T-Bcell interactions with antigen specific, cognate pairs (Bartlett et al.,J. Immunol., 143:1745 (1989); Kupfer et al., J. Exp. Med., 165:1565(1987) and Poo et al., Nature, 332:378 (1988)).

Although terminal B cell differentiation requires both contact- andlymphokine-mediated stimuli from T cells, intermediate stages of B celldifferentiation can be induced by activated T cell surfaces in theabsence of secreted factors (Crow et al., J. Exp. Med., 164:1760 (1986);Brian, Proc. Natl. Acad. Sci., USA, 85:564 (1988); Sekita et al., Eur.J. Immunol., 18:1405 (1988); Hodgkin et al., J. Immunol., 145:2025(1990); Noelle et al., FASEB J, 5:2770 (1991)). These intermediateeffects on B cells include induction of surface CD23 expression (Crow etal., Cell Immunol., 121:94 (1989)), enzymes associated with cell cycleprogression (Pollok et al., J. Immunol., 146:1633 (1991)) andresponsiveness to lymphokines (Noelle et al., FASEB J, 5:2770 (1989);Pollok et al., J. Immunol., 146:1633 (1991)). Recently some of theactivation-induced T cell surface molecules that direct B cellactivation have been identified. Additionally, functional studies havecharacterized some features of activation-induced T cell surfacemolecules that direct B cell activation. First, T cells acquire theability to stimulate B cells 4-8 h following activation (Bartlett etal., J. Immunol., 145:3956 (1990) and Tohma et al., J. Immunol.,146:2544 (1991)). Second, the B cell stimulatory activity associatedwith the surfaces of activated T cells is preserved on paraformaldehydefixed cells (Noelle et al., J. Immunol., 143:1807 (1989); Cros et al.,J. Exp. Med., 164:1760 (1986); Pollok et al., J. Immunol., 146:1633(1991); Tohma et al., J. Immunol., 146:2544 (1991); and Kubota et al.,Immunol., 72:40 (1991)) and on purified membrane fragments (Hodgkin etal., J. Immunol., 145:2025 (1990) and Martinez et al., Nature, 290:60(1981)). Third, the B cell stimulatory activity is sensitive to proteasetreatment (Noelle et al., J. Immunol., 143:1807 (1989); Sekita et al.,Eur. J. Immunol., 18:1405 (1988); and Hodgkin et al., J. Immunol.,145:2025 (1990). Fourth, the process of acquiring these surface activestructures following T cell activation is inhibited by cycloheximide(Tohma et al., J. Immunol., 196:2349 (1991) and Hodgkin et al., J.Immunol., 195:2025 (1990) ).

A cell surface molecule, CD40, has been identified on immature andmature B lymphocytes which, when crosslinked by antibodies, induces Bcell proliferation. Valle et al., Eur. J. Immunol., 19:1463-1467 (1989);Gordon et al., J. Immunol., 140:1425-1430 (1988); Gruder et al., J.Immunol., 142:4144-4152 (1989).

CD40 has been molecularly cloned and characterized (Stamenkovic et al.,EMBO J., 8:1403-1410 (1989)).

CD40 is expressed on B cells, interdigitating dendritic cells,macrophages, follicular dendritic cells, and thymic epithelium (Clark,Tissue Antigens 36:33 (1990); Alderson et al., J. Exp. Med., 178:669(1993); Galy et al., J. Immunol. 142:772 (1992) ). Human CD40 is a typeI membrane protein of 50 kDa and belongs to the nerve growth factorreceptor family (Hollenbaugh et al., Immunol. Rev., 138:23 (1994)).Signaling through CD40 in the presence of IL-10 induces IgA, IgM and IgGproduction, indicating that isotype switching is regulated through theseinteractions. The interaction between CD40 and its ligand results in aprimed state of the B cell, rendering it receptive to subsequentsignals.

Also, a ligand for CD40, gp39 (also called CD40 ligand or CD40L) hasrecently been molecularly cloned and characterized (Armitage et al.,Nature, 357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101(1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)). The gp39protein is expressed on activated, but not resting, CD4⁺ Th cells.Spriggs et al., J. Exp. Med., 176:1543-1550 (1992); Lane et al., Eur. J.Immunol., 22:2573-2578 (1992); and Roy et al., J. Immunol., 151:1-14(1993). Cells transfected with gp39 gene and expressing the gp39 proteinon their surface can trigger B cell proliferation and, together withother stimulatory signals, can induce antibody production. Armitage etal., Nature, 357:80-82 (1992); and Hollenbaugh et al., EMBO J.,11:4313-4319 (1992). In particular, the ligand for CD40, gp39, has beenidentified for the mouse (Noelle et al., Proc. Natl. Acad. Sci. USA,89:6550 (1992); Armitage et al., Nature, 357:80 (1992)) and for humans(Hollenbaugh et al., Embo. J. 11:4313 (1992); Spriggs et al., J. Exp.Met., 176:1543 (1992)). gp39 is a type II membrane protein and is partof a new gene super family which includes TNF-α, TNF-β and the ligandsfor FAS, CD27, CD30 and 4-1BB.

Expression of gp39 can be readily induced in vitro on CD4⁺ T cells usingeither anti-CD3 antibody or phorbol myristate acetate (PMA) plusionomycin. Expression is rapid and transient, peaking at 6-8 hours andreturning to near resting levels between 24 and 48 hours (Roy et al., J.Immunol., 151:2497 (1993)). In vivo, gp39 has been reported in humans tobe present on CD4⁺ T cells in the mantle and centrocytic zones oflymphoid follicles and the periarteriolar lymphocyte sheath of thespleen, in association with CD40⁺ B cells (Lederman et al., J. Immunol.,149:3807 (1992)). gp39⁺ T cells produce IL-2, IL-4 and IFN-γ (Van derEetwegh et al., J. Exp. Med., 178:1555 (1993)).

Unique insights into the novel role of gp39 in the regulation of humoralimmunity have been provided by studies of a human disease, X-linkedhyper-IgM syndrome (HIM). HIM is a profound, X-linked immunodeficiencytypified by a loss in thymus dependent humoral immunity, the inabilityto produce IgG, IgA and IgE. Mutations in the gp39 gene were responsiblefor the expression of a non-functional gp39 protein and the inability ofthe helper T cells from HIM patients to activate B cells (Allen et al.,Science, 259:990 (1993); Aruffo et al., Cell, 72:291 (1993); DiSanto etal., Nature, 361:541 (1993); Korthauer et al., Nature, 361:539 (1993)).These studies support the conclusion that early after T cell receptorengagement of the peptide/MHC class II complex, gp39 is induced on thecognate helper T cell, and the binding of gp39 to CD40 on the B cellinduces the B cell to move into the cell cycle and differentiate toimmunoglobulin (Ig) secretion and isotype switching.

Functional studies have shown that treatment of mice with anti-gp39completely abolished the antibody response against thymus dependentantigens (SRBC and TNP-KLH), but not thymus independent antigens(TNP-Ficoll) (Foy et al., J. Exp. Med., 178:1567 (1993)). In addition,treatment with anti-gp39 prevented the development of collagen-inducedarthritis (CIA) in mice injected with collagen (Durie et al., Science,261:1328 (1993)). Finally, anti-gp39 prevented formation of memory Bcells and germinal centers in mouse spleen (Foy et al., J. Exp. Med.,180:157 (1994)). Collectively, these data provide extensive evidencethat the interaction between gp39 on T cells and CD40 on B cells isessential for antibody responses against thymus dependent antigens.

Recently, a number of murine models of autoimmune disease have beenexploited to evaluate the potential therapeutic value of anti-gp39administration on the development of disease. A brief discussion of theresults of studies in these models are provided below:

Collagen-Induced Arthritis: CIA is an animal model for the humanautoimmune disease rheumatoid arthritis (RA) (Trenthorn et al., J. Exp.Med., 146:857 (1977)). This disease can be induced in many species bythe administration of heterologous type II collagen (Courtenay et al.,Nature, 283:665 (1980); Cathcart et al., Lab. Invest., 54:26 (1986)).

To study the effect anti-gp39 on the induction of CIA (Durie et al.,Science, 261:1328 (1993)) male DBA1/J mice were injected intradermallywith chick type II collagen emulsified in complete Freund's adjuvant atthe base of the tail. A subsequent challenge was carried out 21 dayslater. Mice were then treated with the relevant control antibody oranti-gp39. Groups of mice treated with anti-gp39 showed no titers ofanti-collagen antibodies compared to immunized, untreated control mice.Histological analysis indicated that mice treated with anti-gp39antibody showed no signs of inflammation or any of the typicalpathohistological manifestations of the disease observed in immunizedanimals. These results indicated that gp39-CD40 interactions areabsolutely essential in the induction of CIA. If the initial cognateinteraction between the T cell and B cell is not obtained, then thedownstream processes, such as autoantibody formation and the resultinginflammatory responses, do not occur.

Recently it has been shown that gp39 is important in activatingmonocytes to produce TNF-α and IL-6 in the absence of GM-CSF, IL-3 andIFN-γ (Alderson et al., J. Exp. Med., 178:669 (1993)). TNF-α has beenimplicated in the CIA disease process (Thorbecke et al., Eur. J.Immunol., 89:7375 (1992) and in RA (DiGiovane et al., Ann. Rheum. Dis.,47:68 (1988); Chu et al., Arthrit. Rheum., 39:1125 (1991); Brennan etal., Eur. J. Immunol., 22:1907 (1992). Thus, inhibition of TNF-α byanti-gp39 may have profound anti-inflammatory effects in the joints ofarthritic mice. Both inhibition of TNF-α and of T cell-B cellinteractions by anti-gp39 may be contributory to manifestations of CIA.

Experimental Allergic Encephalomyelitis (EAE): EAE is an experimentalautoimmune disease of the central nervous system (CNS) (Zamvil et al,Ann. Rev. Immunol., 8:579 (1990) and is a disease model for the humanautoimmune condition, multiple sclerosis (MS) (Alvord et al.,"Experimental Allergic Model for Multiple Sclerosis," NY 511 (1984)). Itis readily induced in mammalian species by immunizations of myelin basicprotein purified from the CNS or an encephalitogenic proteolipid (PLP).SJL/J mice are a susceptible strain of mice (H-2^(s)) and, uponinduction of EAE, these mice develop an acute paralytic disease and anacute cellular infiltrate is identifiable within the CNS.

Classen and co-workers (unpublished data) have studied the effects ofanti-gp39 on the induction of EAE in SJL/J mice. They found that EAEdevelopment was completely suppressed in the anti-gp39 treated animals.In addition, anti-PLP antibody responses were delayed and reducedcompared to those obtained for control animals.

EAE is an example of a cell-mediated autoimmune disease mediated via Tcells, with no direct evidence for the requirement for autoantibodies indisease progression. Interference with the interaction between gp39 andCD40 prevents disease induction and the adoptive transfer of disease.

Chronic (c) and acute (a) graft-versus-host-disease (GVHD): Chronic andacute GVHD result from donor cells responding to host disparate MHCalleles. In cGVHD (H-2^(d) →H-2^(bd)), heightened polyclonalimmunoglobulin production is due to the interaction of allospecifichelper T cells and the host B cells. In vivo administration of anti-gp39antibody blocked cGVHD-induced serum anti-DNA autoantibodies, IgEproduction, spontaneous immunoglobulin production in vitro, associatedsplenomegaly and the ability to transfer disease. Durie F. H. et al., J.Clin. Invest., 94:133 (1994). Antibody production remained inhibited forextended periods of time after termination of anti-gp39 administration.Anti-allogeneic cytotoxic T lymphocyte (CTL) responses induced in aGVHDwere also prevented by the in vivo administration of anti-gp39. Thesedata suggest that CD40-gp39 interactions are critical in the generationof both forms of GVHD. The fact that CTL responses were inhibited and abrief treatment with anti-gp39 resulted in long-term prevention ofdisease suggest permanent alterations in the T cell compartment by theco-administration of allogeneic cells and anti-gp39 antibody.

Various research groups have reported the production of murineantibodies specific to gp39, which are disclosed to possess therapeuticutility as immunosuppressants. For example, WO 93/09812, published May27, 1993, and assigned to Columbia University; EP 0,555,880, publishedAug. 18, 1993, and PCT US/94/09872, filed Sep. 2, 1994 by Noelle et aland assigned to Dartmouth College, describe murine antibodies specificto gp39 and their use as therapeutics.

However, while murine antibodies have applicability as therapeuticagents in humans, they are disadvantageous in some respects.Specifically, murine antibodies, because of the fact that they are offoreign species origin, may be immunogenic in humans. This often resultsin a neutralizing antibody response, which is particularly problematicif the antibodies are desired to be administered repeatedly, e.g., intreatment of a chronic or recurrent disease condition. Also, becausethey contain murine constant domains they may not exhibit human effectorfunctions.

In an effort to eliminate or reduce such problems, chimeric antibodieshave been disclosed. Chimeric antibodies contain portions of twodifferent antibodies, typically of two different species. Generally,such antibodies contain human constant and another species, typicallymurine variable regions. For example, some mouse/human chimericantibodies have been reported which exhibit binding characteristics ofthe parental mouse antibody, and effector functions associated with thehuman constant region. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al.,U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No. 4,816,397, allof which are incorporated by reference herein. Generally, these chimericantibodies are constructed by preparing a genomic gene library from DNAextracted from pre-existing murine hybridomas (Nishimura et al., CancerResearch, 47:999 (1987)). The library is then screened for variableregion genes from both heavy and light chains exhibiting the correctantibody fragment rearrangement patterns. Alternatively, cDNA librariesare prepared from RNA extracted from the hybridomas and screened, or thevariable regions are obtained by polymerase chain reaction. The clonedvariable region genes are then ligated into an expression vectorcontaining cloned cassettes of the appropriate heavy or light chainhuman constant region gene. The chimeric genes are then expressed in acell line of choice, usually a murine myeloma line. Such chimericantibodies have been used in human therapy.

In a commonly assigned application, Ser. No. 07/912,292, "Primatized"™antibodies are disclosed which contain human constant and Old Worldmonkey variable regions. These Primatized™ antibodies are well toleratedin humans given their low or weak immunogenicity.

Also, humanized antibodies are known in the art. Ideally, "humanization"results in an antibody that is less immunogenic, with complete retentionof the antigen-binding properties of the original molecule. In order toretain all the antigen-binding properties of the original antibody, thestructure of its combining-site has to be faithfully reproduced in the"humanized" version. This can potentially be achieved by transplantingthe combining site of the nonhuman antibody onto a human framework,either (a) by grafting the entire nonhuman variable domains onto humanconstant regions to generate a chimeric antibody (Morrison et al., Proc.Natl. Acad. Sci., USA, 81:6801 (1984); Morrison and Oi, Adv. Immunol.,44:65 (1988) (which preserves the ligand-binding properties, but whichalso retains the immunogenicity of the nonhuman variable domains); (b)by grafting only the nonhuman CDRs onto human framework and constantregions with or without retention of critical framework residues (Joneset al., Nature, 321:522 (1986); Verhoeyen et al., Science, 239:1539(1988)); or (c) by transplanting the entire nonhuman variable domains(to preserve ligand-binding properties) but also "cloaking" them with ahuman-like surface through judicious replacement of exposed residues (toreduce antigenicity) (Padlan, Molec. Immunol., 28:489 (1991)).

Essentially, humanization by CDR grafting involves transplanting onlythe CDRs onto human fragment onto human framework and constant regions.Theoretically, this should substantially eliminate immunogenicity(except if allotypic or idiotypic differences exist). However, it hasbeen reported that some framework residues of the original antibody alsoneed to be preserved (Riechmann et al., Nature, 332:323 (1988); Queen etal., Proc. Natl. Acad. Sci. USA, 86:10,029 (1989)).

The framework residues which need to be preserved can be identified bycomputer modeling. Alternatively, critical framework residues maypotentially be identified by comparing known antibody combining sitestructures (Padlan, Molec. Immun., 31(3):169-217 (1994)).

The residues which potentially affect antigen binding fall into severalgroups. The first group comprises residues that are contiguous with thecombining site surface which could therefore make direct contact withantigens. They include the amino-terminal residues and those adjacent tothe CDRs. The second group includes residues that could alter thestructure or relative alignment of the CDRs either by contacting theCDRs or the opposite chains. The third group comprises amino acids withburied side chains that could influence the structural integrity of thevariable domains. The residues in these groups are usually found in thesame positions (Padlan, 1994 (Id.) according to the adopted numberingsystem (see Kabat et al., "Sequences of proteins of immunologicalinterest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services,NIH, Bethesda, Md., 1991).

However, while humanized antibodies are desirable because of theirpotential low immunogenicity in humans, their production isunpredictable. For example, sequence modification of antibodies mayresult in substantial or even total loss of antigen binding function, orloss of binding specificity. Alternatively, "humanized antibodies" maystill exhibit immunogenicity in humans, irrespective of sequencemodification.

Thus, there still exists a significant need in the art for novelhumanized antibodies to desired antigens. More specifically, thereexists a need in the art for humanized antibodies specific to gp39,because of their potential as immunotherapeutic agents.

OBJECTS OF THE INVENTION

Toward this end, it is an object of the invention to provide humanizedantibodies which are specific to human gp39.

More specifically, it is an object of the invention to provide humanizedantibodies derived from murine antibodies to gp39 and in particular24-31, a specific murine antibody which binds to human gp39.

It is also an object of the invention to provide pharmaceuticalcompositions containing humanized antibodies which are specific to humangp39.

It is a more specific object of the invention to provide pharmaceuticalcompositions containing humanized antibodies derived from 24-31, amurine antibody which specifically binds to human gp39.

It is another specific object of the invention to provide methods ofusing humanized antibodies to human gp39 for treatment of human diseaseconditions, which are treatable by modulation of gp39 expression and/orinhibition of the gp39/CD40 binding interaction including, e.g.,autoimmune diseases such as systemic lupus erythematosus, rheumatoidarthritis, multiple sclerosis, idiopathic thrombocytopenic purpura(ITP), diabetes and non-autoimmune conditions such as graft-versus-hostdisease and transplantation.

It is still another object of the invention to provide nucleic acidsequences which encode for humanized antibodies to human gp39.

It is a more specific object of the invention to provide nucleic acidsequences which encode humanized antibodies derived from 24-31, a murineantibody which specifically binds to human gp39 antigen.

It is another object of the invention to provide vectors which providefor the expression of humanized antibodies to human gp39, in particularhumanized antibodies derived from 24-31, a murine antibody whichspecifically binds to human gp39 antigen.

SUMMARY OF THE INVENTION

In its broadest embodiment, the present invention is directed tohumanized antibodies which retain not less than about one-tenth and morepreferably not lower than one-third the gp39 antigen binding affinity ofthe murine 24-31 antibody and/or which retain not less than aboutone-tenth and more preferably not less than about one-third the in vitrofunctional activity of the murine antibody 24-31, e.g., in B-cell assayswhich measure T-cell dependent antibody production. More particularly,the present humanized antibodies retain at least one-tenth and morepreferably at least about one-third the half-maximal potency in in vitrofunctional activity in a B cell assay at a concentration of not morethan three times the concentration of the 24-31 antibody.

The present invention is further directed to humanized antibodies whichbind to the same epitope as the murine 24-31 antibody and/or which arecapable of competing with the murine 24-31 antibody for inhibiting thebinding of CD40 to gp39 and/or which contain the CDR's of the 24-31antibody.

The present invention is more preferably directed to humanizedantibodies derived from murine 24-31 which possess the humanizedvariable light sequences [SEQ ID NOS.:1-4] and/or humanized variableheavy sequences set forth below:

    (1) DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT                  -     GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC QQYNSYPYT FGGGTKLEIK;                  - (2) DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT                                                   -     GVPDRFSGSGSGTDFTLTISSLQAEDV                                           ADYFC QQYNSYPYT FGGGTKLEIK;                                                     - (3) DIVMTQSPSFMSTSVGDRVTITC                                               KASQNVITAVA WYQQKPGKSPKLLIY                                                   SASNRYT                                   -     GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC QQYNSYPYT FGGGTKLEIK;                  - (4) DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT                                                   -     GVPDRFSGSGSGTDFTLTISSMQAEDV                                           ADYFC QQYNSYPYT FGGGTKLEIK         

and a humanized variable heavy sequence [SEQ ID NOS.:5-8] selected fromthe following group:

    (1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPSLKS                                                    -     RISISRDTSKNQFSLKLSSVTA                                                ADTGVYYCAC RSYGRTPYYFDF                                                       WGQGTTLTVSS;                         - (2) EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPS                                                LKS                                  -     RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS;                                                         - (3) EVQLQESGPGLVKPSQTLSLTC                                                AVSGDSIT NGFWI WIRKHPGNKLEYMG                                                 YISYSGSTYYNPSLKS                     -     RISISRDTSNNQFSLNLNSVTRADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS;                                                         - (4) EVQLQESGPGLVKPSETLSLTC                                                AVYGDSIT NGFWI WIRKPPGNKLEYMG                                                 YISYSGSTYYNPSLKS                     -     RISISRDTSKNQFYLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS        

as well as variants and equivalents thereof. Variants and equivalentsthereof in the present invention are intended to embrace humanizedimmunoglobulin sequences wherein one or several of the amino acidresidues in the above identified humanized variable heavy and/orvariable light sequences are modified by substitution, addition and/ordeletion in such manner that does not substantially effect gp39 antigenbinding affinity. In particular, the present invention embraces variantsand equivalents which contain conservative substitution mutations, i.e.,the substitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid within the same general class, e.g., an acidic amino acid, ora basic amino acid, a neutral amino acid by another amino acid withinthe same class. What is intended by a conservative amino acidsubstitution is well known in the art. Preferably, such variants andequivalents will retain not less than about one-tenth and morepreferably not less than about one-third the gp39 antigen bindingaffinity as the parent murine 24-31 antibody and more preferably notless than about one-third the gp39 antigen binding affinity as themurine 24-31 antibody. Additionally, such variants and equivalents willpreferably retain not lower than one-tenth and more preferably retain atleast about one-third the in vitro functional activity of murineantibody 24-31, e.g., in B-cell assays which measure T-cell dependentantibody production. More preferably, these variants and equivalentswill retain at least about one-third the in vitro functional activity ofmurine antibody 24-31, for example, in B-cell assays which measureT-cell dependent antibody production. More specifically, theseantibodies will retain the half-maximal potency in in vitro functionalactivity in a B cell assay at a concentration of not more than aboutthree times the concentration of the parent 24-31 antibody.

The present invention is further directed to nucleic acid sequenceswhich encode for the expression of such humanized antibodies, as well asexpression vectors which provide for the production of humanizedantibodies in recombinant host cells. In the most preferred embodimentsthese DNA sequences will encode for the humanized variable heavy and/orhumanized variable light sequences [SEQ ID NOS.:1-4] set forth below:

    (1) DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT                  -     GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC QQYNSYPYT FGGGTKLEIK;                  - (2) DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT                                                   -     GVPDRFSGSGSGTDFTLTISSLQAEDV                                           ADYFC QQYNSYPYT FGGGTKLEIK;                                                     - (3) DIVMTQSPSFMSTSVGDRVTITC                                               KASQNVITAVA WYQQKPGKSPKLLIY                                                   SASNRYT                                   -     GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC QQYNSYPYT FGGGTKLEIK;                  - (4) DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT                                                   -     GVPDRFSGSGSGTDFTLTISSMQAEDV                                           ADYFC QQYNSYPYT FGGGTKLEIK         

and a humanized variable heavy sequence [SEQ ID NOS.:5-8] selected fromthe following group:

    (1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPSLKS                                                    -     RISISRDTSKNQFSLKLSSVTA                                                ADTGVYYCAC RSYGRTPYYFDF                                                       WGQGTTLTVSS;                         - (2) EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPS                                                LKS                                  -     RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS;                                                         - (3) EVQLQESGPGLVKPSQTLSLTC                                                AVSGDSIT NGFWI WIRKHPGNKLEYMG                                                 YISYSGSTYYNPSLKS                     -     RISISRDTSNNQFSLNLNSVTRADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS;                                                         - (4) EVQLQESGPGLVKPSETLSLTC                                                AVYGDSIT NGFWI WIRKPPGNKLEYMG                                                 YISYSGSTYYNPSLKS                     -     RISISRDTSKNQFYLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS.       

Moreover, the present invention also embraces equivalent and variantsthereof as defined supra.

The present invention is further directed to the use of theabove-identified humanized antibodies specific to gp39 aspharmaceuticals. The present invention is also directed to the use ofthe subject humanized anti-gp39 antibodies for treating diseasestreatable by modulation of gp39 expression or by inhibition of thegp39/CD40 interaction. The present invention is more particularlydirected to the use of humanized antibodies of the above-identifiedhumanized antibodies specific to gp39 for the treatment of autoimmunedisorders, for example, rheumatoid arthritis, multiple sclerosis,diabetes, systemic lupus erythematosus and ITP. The present invention isfurther directed to the use of the subject humanized antibodies to gp39for the treatment of non-autoimmune disorders includinggraft-versus-host disease and for inhibiting graft rejection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the IDEC expression vector N5KGl used to expresshumanized and chimeric antibodies derived from 24-31.

FIG. 2a contains results of a B cell proliferation assay which contactshuman PBLs with soluble gp39-CD8, recombinant human IL-4 and the murine24-31 antibody or control murine IgGl monoclonal antibody whichdemonstrate that 24-31 antibody inhibits B cell proliferation induced bygp39.

FIG. 2B contains results of B cell differentiation assay using mitomycintreated T cells activated with immobilized anti-CD3 cultured in thepresent of IGD⁺ B cells and different concentrations of the 24-31antibody which demonstrate that 24-31 antibody inhibits T-cell dependentpolyclonal antibody production by human B cells.

FIG. 3 contains FACS of non-transfected CHO cells and a gp39transfectant.

FIG. 4 [SEQ ID NO.:24] contains the amino acid sequence and DNA sequencecorresponding to a preferred humanized variable light sequence(including the complementarity determining regions) referred to as VL#1or preferred humanized variable light sequence (1).

FIG. 5 [SEQ ID NO.:25] contains the amino acid and DNA sequencecorresponding to a preferred humanized variable ligand sequence(including the complementarity determining regions) referred to as VL#2or preferred humanized variable light sequence (2).

FIG. 6 [SEQ ID NO.:26] contains the amino acid and DNA sequencecorresponding to a preferred humanized variable heavy sequence(including the complementarity determining regions) referred to as VH#1of preferred humanized variable heavy sequence (1).

FIG. 7 [SEQ ID NO.:27] contains the amino acid and DNA sequence of thevariable light sequence of 24-31 (non-humanized).

FIG. 8 [SEQ ID NO.:28] contains the amino acid and DNA sequence of thevariable heavy sequence of 24-31 (non-humanized).

FIG. 9 compares binding of murine 24-31, chimeric 24-31 and a humanized24-31 antibody to gp39 expressing CHO cells.

FIG. 10 contains results of a competition assay comparing the binding of24-31 (biotin) and humanized, chimeric and 24-31 to gp39 expressing CHOcells.

FIG. 11 contains results of an assay which measures effects of murine24-31 and a humanized 24-31 antibody of the invention on human IgMproduction by B cells cultured in the presence of mitomycin C treated Tcells.

FIG. 12 contains results of an assay comparing binding of two humanizedantibodies of the present invention to gp39 expressing CHO cells.

FIG. 13 contains the Scatchard plot for murine 24-31.

FIG. 14 contains the Scatchard plot for humanized Version 1.

FIG. 15 contains the Scatchard plot for humanized Version 2.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, definitions of certain terms whichare used in this disclosure are set forth below:

Humanized Antibody

This will refer to an antibody derived from a non-human antibody,typically murine, that retains or substantially retains theantigen-binding properties of the parent antibody but which is lessimmunogenic in humans. This may be achieved by various methods including(a) grafting the entire non-human variable domains onto human constantregions to generate chimeric antibodies, (b) grafting only the non-humanCDRs onto human framework and constant regions with or without retentionof critical framework residues, or (c) transplanting the entirenon-human variable domains, but "cloaking" them with a human-likesection by replacement of surface residues. Such methods are disclosedin Jones et al., Morrison et al., Proc. Natl. Acad. Sci., 81:6851-6855(1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen etal., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498(1991); Padlan, Molec. Immun., 31(3):169-217 (1994), all of which areincorporated by reference.

Complementarity Determining Region, or CDR

The term CDR, as used herein, refers to amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native immunoglobulin binding site as delineated by Kabat etal (1991).

Framework Region

The term FR, as used herein, refers to amino acid sequences interposedbetween CDRs. These portions of the antibody serve to hold the CDRs inappropriate orientation (allows for CDRs to bind antigen).

Constant Region

The portion of the antibody molecule which confers effector functions.In the present invention, murine constant regions are substituted byhuman constant regions. The constant regions of the subject chimeric orhumanized antibodies are derived from human immunoglobulins. The heavychain constant region can be selected from any of the five isotypes:alpha, delta, epsilon, gamma or mu. Further, heavy chains of varioussubclasses (such as the IgG subclasses of heavy chains) are responsiblefor different effector functions and thus, by choosing the desired heavychain constant region, chimeric antibodies with desired effectorfunction can be produced. Preferred constant regions are gamma 1 (IgGl),gamma 3 (IgG3) and gamma 4 (IgG4). More preferred is an Fc region of thegamma 1 (IgG1) isotype. The light chain constant region can be of thekappa or lambda type, preferably of the kappa type.

Chimeric Antibody

This is an antibody containing sequences derived from two differentantibodies, which typically are of different species. Most typicallychimeric antibodies comprise human and murine antibody fragments,generally human constant and murine variable regions.

Immunogenicity

A measure of the ability of a targeting protein or therapeutic moiety toelicit an immune response (humoral or cellular) when administered to arecipient. The present invention is concerned with the immunogenicity ofthe subject humanized antibodies or fragments thereof.

Humanized or Chimeric Antibody of Reduced Immuno-Genicity

This refers to an antibody or humanized antibody exhibiting reducedimmunogenicity relative to the parent antibody, e.g., the 24-31antibody.

Humanized Antibody Substantially Retaining the Binding Properties of theParent Antibody

This refers to a humanized or chimeric antibody which retains theability to specifically bind the antigen recognized by the parentantibody used to produce such humanized or chimeric antibody. Humanizedor chimeric antibodies which substantially retain the binding propertiesof 24-31 will bind to human gp39. Preferably the humanized or chimericantibody will exhibit the same or substantially the same antigen-bindingaffinity and avidity as the parent antibody. Ideally, the affinity ofthe antibody will not be less than 10% of the parent antibody affinity,more preferably not less than about 30%, and most preferably theaffinity will not be less than 50% of the parent antibody. Methods forassaying antigen-binding affinity are well known in the art and includehalf-maximal binding assays, competition assays, and Scatchard analysis.Suitable antigen binding assays are described in this application.

The present invention is directed to novel humanized monoclonalantibodies which bind human gp39 and their use as therapeutic agents.The present invention is further directed toward nucleic acid sequenceswhich encode said humanized antibodies, and their expression inrecombinant host cells.

More specifically, the present invention is directed toward humanizedantibodies derived from murine antibody 24-31 which specifically bindsto human gp39.

Murine antibody 24-31 is a murine antibody raised against human gp39which functionally inactivates gp39 both in vitro and in vivo.Therefore, it possesses properties which render it potentially usefulfor treatment of diseases wherein gp39 inactivation and/or modulation orinhibition of the gp39/CD40 interation is desirable. In particular, suchdiseases include autoimmune diseases such as, e.g., rheumatoidarthritis, multiple sclerosis, ITP, diabetes, and systemic lupuserythematosus as well as non-autoimmune diseases such asgraft-versus-host disease and graft rejection.

However, while murine antibody 24-31 possesses functional propertieswhich render it potentially suitable as a therapeutic agent, itpossesses several potential disadvantages. Namely, because it is ofmurine origin it potentially will be immunogenic in humans. Also,because it contains murine constant sequences, it will likely notexhibit the full range of human effector functions and will probably bemore rapidly cleared if administered to humans.

While such disadvantages should not be problematic in the treatment ofsome disease conditions or persons, they pose substantial concern if thedisease treated is of a chronic or recurrent nature. Examples ofrecurrent or chronic diseases include, e.g., autoimmune diseases,wherein the host continually or chronically exhibits an autoimmunereaction against self-antigens.

Therefore, in order to alleviate the disadvantages associated withmurine antibody 24-31, namely potential immunogenicity in humans anddecrease of human effector functions, the present inventors desired toproduce improved, humanized derivatives of the murine 24-31 antibody.While this was the goal of the present invention, the desired result wasnot of a routine or predictable nature. Humanization of antibodiesrequires the careful selection of amino acid residues which are to bemodified, and the judicious selection of residues which are to besubstituted therefor. This is because modification of antibody variableregions, even those involving a few amino acid residues, may causesubstantial deleterious effects on antigen binding. For example,humanized antibodies may exhibit substantially reduced antigen affinityand/or antigen-specificity in relation to the parent antibody.

As noted supra, different methods of humanization of antibodies,including murine antibodies have been reported in the literature. See,e.g., Padlan, Molec. Immunol., 31(3) :169-217 (1994); Padlan, Molec.Immunol., 28:484-498 (1991); Morrison and Oi, Adv. Immunol., 44:65-92(1988), all of which references are incorporated by reference in theirentirety herein. These methods include in particular humanization by CDRgrafting (Jones et al., Nature, 321:522-525 (1986); Verhoeyen et al.,Science, 239:1534-1539 (1988); and the more tailored approach of Padlan,Molec. Immunol., 28:489 (1991) and Padlan, Molec. Immunol., 31:169(1994) which involves the selection of non-essential framework aminoacid residues and their modification by appropriate substitutionmutation. These references are incorporated by reference in theirentirety herein.

As noted, CDR grafting techniques, while successful in some instances,may substantially adversely affect the affinity of the resultanthumanized antibodies. This is believed to occur because some frameworkresidues affect or are essential for and at least affect antigenbinding. Our technique; Padlan (1994) (Id.)) is more refined because weretain only those murine framework residues which we deem critical tothe preservation of the antibody combining site while keeping thesurface properties of the molecule as human as possible. Accordingly,this technique has the potential of producing humanized antibodies whichretain the antigen-binding characteristics of the parent antibody.Because of this, this technique was selected by the present inventors asthe means by which humanized antibodies derived from murine antibody24-31 specific to human gp39 would potentially be obtained.

The cloning of the variable regions of 24-31 (described in detail in theexamples infra) resulted in the identification of the V_(L) and V_(H)sequences utilized by the 24-31 antibody respectively shown in FIG. 7and FIG. 8. After sequencing, the variable regions were then humanized.As noted, this was effected substantially according to the method ofPadlan (1994) (Id.), incorporated by reference supra.

This method generally comprises replacement of the non-human frameworkby human framework residues, while retaining only those frameworkresidues that we deem critical to the preservation of antigen bindingproperties.

Ideally, this methodology will confer a human-like character on thesurface of the xenogeneic antibody thus rendering it less immunogenicwhile retaining the interior and contacting residues which affect itsantigen-binding properties.

More specifically, the 24-31 V_(K) and V_(H) sequences set forth inFIGS. 7 and 8 were humanized by comparison to human antibodies ofreported sequence, which are referred to as "templates."

Specifically, the 24-31 V_(K) was humanized using as templates:

(a) For VL#1, the human V-Kappa subgroup I sequences, e.g., DEN and thelike, as well as the germline 012 (see Cox et al., Eur. J. Immunol.24:827-836 (1994)), and for VL#2, the human V-Kappa subgroup IVsequences, e.g., LEN. Such template sequences are known and are reportedin Kabat is et al. (1991) (Id.) or GenBank.

The 24-31 V_(H) #1 was humanized using as templates

(a) the human V_(H) subgroup IV sequence, 58p2 and

(b) (GenBank Accession No.) Z18320 and the germline 3d75d (S. van derMaarel et al., J. Immunol., 150:2858-2868 (1993).

Such template variable heavy antibody sequences are also known and arereported in Kabat et al., "Sequences of Proteins of ImmunologicalInterest," 5th Ed., NIH (1991) and in GenBank.

The template human variable heavy and light sequences were selectedbased on a number of different criteria, including, in particular, highdegree of sequence similarity with 24-31 overall, as well as similarityin the "important" residues, i.e., those which are believed to becomprised in the V_(L) :V_(H) interface; those which are in contact withthe complementarity determining regions, or which are inwardly pointing.Also, the templates were selected so as to potentially preserve theelectrostatic charge of the 24-31 F_(v) as much as possible, and also soas to preserve glycines, prolines and other specific amino acid residueswhich are believed to affect antigen binding.

This methodology results in the following preferred humanized V_(L) andV_(H) heavy sequences derived from the 24-31 antibody which are setforth below in Table 1 [SEQ ID NOS.:1-4] and Table 2[SEQ ID NOS.:5-8] .As discussed above, the invention further embraces equivalents andvariants of these preferred humanized sequence, e.g., those containingone or more conservative amino acid substitution which do notsubstantially affect gp39 binding. The complementarity determiningregions are identified in FIGS. 7 and 8 which contain the entirevariable heavy and light chain CDR sequences of the parent(non-humanized) 24-31 antibody.

                                      TABLE 1                                     __________________________________________________________________________    HUMANIZED 24-31 VL SEQUENCES                                                  __________________________________________________________________________                   10        20                    40                    60                 70        80                                                          24-31 DIVMTQSQKFMSTSVGDRVSITC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT                GVPDRFSGSGSGTDFTLTISNMQSEDLADYFC                                            -               100                                                           FGGGTKLEIK QQYNSYPYT                                                          - (1) DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT               GVPDRFSGSGSGTDFTLTISSLQPEDFADYFC                                            -  QQYNSYPYT FGGGTKLEIK                                                       - (2) DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT               GVPDRFSGSGSGTDFTLTISSLQAEDVADYFC                                            -  QQYNSYPYT FGGGTKLEIK                                                       - (3) DIVMTQSPSFMSTSVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRT                GVPDRFSGSGSGTDFTLTISSMQPEDFADYFC                                            -  QQYNSYPYT FGGGTKLEIK                                                       - (4) DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT               GVPDRFSGSGSGTDFTLTISSMQAEDVADYFC                                            -  QQYNSYPYT FGGGTKLEIK                                                    __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    HUMANIZED 24-31 VH SEQUENCES                                                  __________________________________________________________________________                 10        20        30          40                                 24-31 EVQLQESGPSLVKPSQTLSLTCSVTGDSIT NGFWI WIRKFPGNKLEYMG YISYSGSTYYNPSL        KS                                                                           -      70          82abc      90                        110                   RISITRDTSQNQFYLQLNSVTTEDTGTYYCAC RSYGRTPYYFDF WGQGTTLTVSS                     - (1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPS        LKS                                                                          -  RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS                  - (2) EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPS        LKS                                                                          -  RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS                  - (3) EVQLQESGPGLVKPSQTLSLTCAVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPS        LKS                                                                          -  RISISRDTSNNQFSLNLNSVTRADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS                  - (4) EVQLQESGPGLVKPSETLSLTCAVYGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPS        LKS                                                                          -  RISISRDTSKNQFYLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS               __________________________________________________________________________

As can be seen therefrom, four preferred humanized framework sequenceswere designed for both the V_(H) and V_(L) chains. Therefore, there are16 different possible humanized 24-31 antibodies which may besynthesized using the above-identified humanized V_(H) and V_(L) 24-31sequences, excluding variants and equivalents containing conservativemodifications.

Humanized 24-31 antibodies containing these humanized variable heavy andlight sequences may be obtained by recombinant methods. It is expectedthat humanized sequences which contain any combination of the abovepreferred humanized variable sequences will result in humanizedantibodies which bind human gp39. Moreover, based on these sequences,the order of preference using the numbering set forth in Table 1 andTable 2 is expected to be as follows:

(1) #1 V_(L) with #1 V_(H) (Version 1)

(2) #2 V_(L) with #1 V_(H) (Version 2)

(3) #1 V_(L) with #2 V_(H) (Version 3)

(4) #2 V_(L) with #2 V_(H) (Version 4)

The above-identified humanized V_(H) and V_(L) sequences may be furthermodified, e.g., by the introduction of one or more additionalsubstitution modifications and also by the addition of other aminoacids. Additional modifications will be selected which do not adverselyaffect antigen (gp39) binding. For example, the inventors contemplatefurther modification of the V_(H) chain by substitution of one or moreof residues 34, 43, 44 and 68 (according to Kabat numbering scheme)Kabat et al. (1991) (Id.). Also, the inventors contemplate modificationof residue 85 of the V_(L) chain. Based on the structural features ofthe antibody combining site, it is believed that modification of suchresidues should also not adversely impact antigen binding.

Moreover, it is expected that the introduction of one or moreconservative amino acid substitutions should not adversely affect gp39binding.

So as to better describe the subject humanized 24-31, V_(H) and V_(L)sequences, the preferred humanized framework sequences [SEQ ID NO.:9-21]are also set forth in Table 3 below, which compares these sequences tothe template human variable heavy and light framework sequences, i.e.,human DEN VK1, Human o12/V36 germline, human LEN VKIV, human 58p2, humanZ18320, and human 3d75d as well as to the unhumanized murine 24-31 V_(H)and V_(L) framework sequences.

                                      TABLE 3                                     __________________________________________________________________________    VK Framework Region Comparisons - Humanized Anti-gp39                         __________________________________________________________________________                  FR1                 FR2                                           Human 012/V3b germline    DIQMTQSPSFLSASVGDRVTITC     WYQQKPGKAPKLLIY                                          Human DEN VKI                               T-------------                                                                E---V---                                                                       Murine 24-31                                                                 V----QK-M-T------S---                                                         QS------                                                                       Padlan VL#1 humanized                                                        V--------------------                                                         S------                                                                         -                                    FR3                                                                     FR4                                           Human 012/V3b    GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC                             Human DEN VK1                                                                E---------SD-------       FGQGTKLEIK                                           Murine 24-31                                                                 D----------------NM-SE-L-D-F-                                                 G-------                                                                       Padlan VL#1                                                                  D------------------------D-F-                                                 G-------                                                                        -                                          FR1                                                               FR2                                           Human LEN VKIV            DIVMTQSPDSLAVSLGERATINC     WYQQKPGQPPKLLIY                                          Murine 24-31                                QKFMST-V-D-VS-T-                                                              S------                                                                        Padlan VL#2 humanized                                                        S------                                                                         -                                    FR3                                                                     FR4                                           Human LEN VKIV      GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC  FGQGTKLEIK                                               Murine 24-31                                NM-S--L-D-F-                                                                  G-------                                                                       Padlan VL#2                                                                  D-F-                                                                          G-------                                                                        -                                          FR1                               Huinan 58p2               QVQLQESGPGLVKPSETLSLTCTVSGGSIS                      Murine 24-31              E---------S----Q------S-T-D--T                      Padlan VH#1 humanized E-------------------------D--T                          Human Z18320 GenBank                                                         Q--------------                                                                Human 3d75d germline                                                         Q--------------                                                                Padlan VH#2 humanized  E--------------Q----------D--T                          -                                FR2               FR3                       Human 58p2        WIRQPPGKGLEWIG    RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR                                           Murine 24-31                                KF--NK--YM-                                                                   IS-TR---Q---Y-Q-N---TE--GT----C                                                Padlan VH#1                                                                  K---NK--YM-                                                                   IS--R-------------------G-----C                                                Human Z18320                                                                 A--------                                                                      Human 3d75d                                                                  H---------                                                                     Padlan VH#2                                                                  KH--NK--YM-                                                                   IS--R-------------------G-----C                                                 - Human 58p2       WGQGTMVTVSS                                               Murine 24-31                                                                 TL----                                                                         Padlan VH#1                                                                  TL----                                                                         Hutnan Z18320                                                                 Padlan VH#2                                                                  TL----                                                                       __________________________________________________________________________

In order to produce humanized antibodies, DNA sequences are synthesizedwhich encode for the afore-identified humanized V_(L) and V_(H)sequences. As noted, taking into account these four humanized V_(L)sequences, and four humanized V_(H) sequences, there are 16 potentialhumanized antigen combining sites which may be synthesized. Also, thereare even more potential humanized antigen combining sites taking intoaccount the potential substitution of residues 34, 43, 44 and 68 of thehumanized V_(H) and residue 85 of the humanized V_(L) by other aminoacid residues and/or the potential incorporation of conservativesubstitution mutations. Two of the preferred humanized variable lightsequences (1) and (2) and a preferred humanized variable heavy sequence(1) including the complementarity determining regions and correspondingDNA sequences [SEQ ID NOS.:24-26] are set forth in FIGS. 4, 5 and 6 [SEQID NOS.:24-26], respectively.

Methods for synthesizing DNA encoding for a protein of known sequenceare well known in the art. Using such methods, DNA sequences whichencode the subject humanized V_(L) and V_(H) sequences are synthesized,and then expressed in vector systems suitable for expression ofrecombinant antibodies. This may be effected in any vector system whichprovides for the subject humanized V_(L) and V_(H) sequences to beexpressed as a fusion protein with human constant domain sequences andassociate to produce functional (antigen binding) antibodies.

Expression vectors and host cells suitable for expression of recombinantantibodies and humanized antibodies in particular, are well known in theart.

The following references are representative of methods and vectorssuitable for expression of recombinant immunoglobulins which areincorporated by reference herein: Weidle et al., Gene, 51:21-29 (1987);Dorai et al., J. Immunol., 13(12):4232-4241 (1987); De Waele et al.,Eur. J. Biochem., 176:287-295 (1988); Colcher et al., Cancer Res.,49:1738-1745 (1989); Wood et al., J. Immunol., 145(a):3011-3016 (1990);Bulens et al., Eur. J. Biochem., 195:235-242 (1991); Beggington et al.,Biol. Technology, 10:169 (1992); King et al., Biochem. J., 281:317-323(1992); Page et al., Biol. Technology, 9:64 (1991); King et al.,Biochem. J., 290:723-729 (1993); Chaudary et al., Nature, 339:394-397(1989); Jones et al., Nature, 321:522-525 (1986); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Benhar et al., Proc. Natl. Acad. Sci. USA,91:12051-12055 (1994); Singer et al., J. Immunol., 150:2844-2857 (1993);Cooto et al., Hybridoma, 13(3):215-219 (1994); Queen et al., Proc. Natl.Acad. Sci. USA, 86:10029-10033 (1989); Caron et al., Cancer Res.,32:6761-6767 (1992); Cotoma et al., J. Immunol. Meth., 152:89-109(1992). Moreover, vectors suitable for expression of recombinantantibodies are commercially available.

Host cells known to be capable of expressing functional immunoglobulinsinclude by way of example mammalian cells such as Chinese Hamster Ovary(CHO) cells, COS cells, myeloma cells, bacteria such as Escherichiacoli, yeast cells such as Saccharomyces cerevisiae, among other hostcells. Of these, CHO cells are used by many researchers given theirability to effectively express and secrete immunoglobulins.

Essentially, recombinant expression of humanized antibodies are effectedby one of two general methods. In the first method, the host cells aretransfected with a single vector which provides for the expression ofboth heavy and light variable sequences fused to selected constantregions. In the second method, host cells are transfected with twovectors, which respectively provide for expression of either thevariable heavy or light sequence fused to selected constant regions.

Human constant domain sequences are well known in the art, and have beenreported in the literature. Preferred human V_(L) sequences includes theKappa and lambda constant light sequences. Preferred human heavyconstant sequences include human gamma 1, human gamma 2, human gamma 3,human gamma 4 and mutated versions thereof which provide for alteredeffect or function, e.g. enhanced in vivo half-life and reduced Fcreceptor binding.

Preferred modifications of the human gamma 4 constant domain include Pand/or E modifications, which respectively refer to the change of aleucine to a glutamic acid at position 236 and/or the change of a serineto a proline (Kabat numbering) at position 229 such as described incommonly assigned Attorney Docket No. 012712-165 filed on Sep. 6, 1995and incorporated by reference in its entirety herein.

A particularly preferred vector system comprises the expression vectorsdescribed in commonly assigned U.S. Ser. No. 08/476,237 filed Jun. 7,1995, Ser. No. 08/397,072, filed Jan. 25, 1995 and Ser. No. 07/912,122filed Jul. 10, 1992, Ser. No. 07/886,281 filed Mar. 23, 1992, and Ser.No. 07/735,064 filed Jul. 25, 1991, all incorporated by reference intheir entirety.

In particular, these applications describe vector systems for theproduction of recombinant antibodies, referred to as TCAE 5.2 and TCAE 6which comprise the following:

1) Four transcriptional cassettes in tandem order:

(a) a human immunoglobulin light chain constant region. In TCAE 5.2 thisis the human immunoglobulin Kappa light chain constant region (Kabatnumbering amino acids 108-214, allotype Km 3) and in TCAE 6 the humanimmunoglobulin light chain lambda constant region (Kabat numbering aminoacids 108-215, genotype Oz minus, Mcg minus, Ke minus allotype).

(b) a human immunoglobulin heavy chain constant region; in bothconstructs the human immunoglobulin heavy chain is a gamma/constantregion (Kabat numbering amino acids 114-478 allotype Gm1a, Gm12).

(c) DHFR; containing its own eukaryotic promoter and polyadenylationregion; and

(d) NEO; also containing its own eukaryotic promoter and polyadenylationregion.

2) The human immunoglobulin light and heavy chain cassettes containsynthetic signal sequences for secretion of the immunoglobulin chains;and

3) The human immunoglobulin light and heavy chain cassettes containspecific DNA links which allow for the insertion of light and heavyimmunoglobulin variable regions which maintain the translational readingframe and do not alter the amino acids normally found in immunoglobulinchains.

These vectors are preferably utilized in CHO cells. The subjectantibodies are preferably expressed in the above-described vectorsystems.

However, the subject humanized antibody sequences derived from thenumber 24-31 antibody may be expressed in any vector system whichprovides for the expression of functional antibodies, i.e., those whichbind gp39 antigen.

In particular, the inventors elected to express the subject humanizedV_(L) and V_(H) sequences, as well as the native (unmodified) V_(L) andV_(H) sequences derived from 24-31 in CHO cells using the N5KGlexpression vector which contains human Kappa and human gamma 1 constantregions. The N5KG1 expression vector is depicted schematically inFIG. 1. As hoped, the chimeric antibody derived from 24-31, whenexpressed in CHO cells binds gp39 (by demonstrated binding to CHO-gp39transfectant). Also, several humanized antibodies of the inventionderived from 24-31 when expressed using this vector system resulted infunctional (gp39 binding) antibodies.

The present invention is further described through presentation of thefollowing examples. These examples are offered by way of illustrationand not by way of limitation.

EXAMPLE 1

Selection of 24-31 Antibody for Humanization

Accumulating evidence in animal models indicates that anti-gp39administration prevents a variety of autoimmune processes and interfereswith allograft rejection. These results provide compelling evidence thatantibodies to human gp39 may have significant therapeutic value in themanagement of autoimmune disease and the transplantation of allogeneictissue and organs in humans. A monoclonal antibody (mAb) specific forhuman gp39 has been reported (Lederman et al., J. Immunol., 199:3817(1992)), and its functional activity in blocking gp39-CD40 interactionsin vitro has been evaluated. To gain greater insights into thefunctional impact of anti-gp39 antibodies on the human immune system, apanel of anti-human gp39 mAbs was generated. From this panel, one mAbappeared superior and was extensively tested for functional inactivationof gp39 in vitro and in vivo.

More specifically, a panel of 6 murine (all IgGl) anti-gp39 antibodieswas generated by immunization with a soluble fusion protein of humangp39 (gp39-CD8), followed by challenge with activated human peripheralblood T cells. Flow cytometric analysis of human peripheral blood Tcells demonstrated that the mAbs recognized a cell surface moleculeexpressed on activated (PMA/ionomycin), but not resting, CD3+T cells,and that the pattern of reactivity was similar to that seen with arecombinant CD40 fusion protein (CD40-Ig) (data not shown).Immunoprecipitation of [³⁵ S] metabolically labeled activated humanperipheral blood T cells revealed that each of the 6 mAbs precipitated amolecule of similar size (33 kDa) to that precipitated by CD40-Ig.Finally, binding of CD40-Ig to gp39 was blocked in the presence of theantibodies indicating recognition of the same molecule, furtherconfirming their specificity. Although all 6 mAbs were capable ofblocking gp39 function, one mAb, 24-31, was selected for extensiveanalysis.

EXAMPLE 2

T Cell-Dependent B Cell Proliferation and Differentiation (IgProduction) is Blocked by Anti-gp39

A number of studies have provided evidence that signals deliveredthrough CD40 by its ligand, gp39, induce B cell activation,proliferation, differentiation, and isotype switching. To determine ifthe anti-gp39 24-31 mAb blocked gp39 function, B cells were culturedwith a soluble fusion protein of gp39 (gp39-CD8) in the presence orabsence of 24-31, and the B cell proliferative response was assessed by³ H-thymidine incorporation. The results, shown in FIG. 2A, demonstratethat gp39-CD8 induced vigorous proliferation of B cells. The presence ofanti-gp39 24-31 mAb completely ablated B cell proliferation induced bygp39-CD8 at concentrations as low as 2.5 μg/ml. To determine whether24-31 interfered with T cell-induced B cell differentiation, B cellswere co-cultured with anti-CD3 activated T cells in the presence orabsence of 24-31. Polyclonal IgM, IgG, and IgA production was assessedafter 12 days. As shown in FIG. 2B, the addition of 24-31 inhibitedpolyclonal IgM, IgG, and IgA antibody production (90-99%). These resultsconfirm previous reports establishing the requirement for gp39-CD40interactions in T cell-dependent B cell differentiation (Nishioka etal., J. Immunol., 153:1027 (1994), and further demonstrate the use ofnewly characterized anti-human gp39 24-31 mAb in blocking gp39 function.

EXAMPLE 3

Anti-gp39 Blocks in Vivo Tetanus Toxoid Specific Antibody Production inScid Mice Reconstituted with Human PBL.

Numerous studies have established that the human immune system can bestudied in vivo under experimental conditions through the use of severecombined immunodeficiency (scid) mice engrafted with human peripheralblood lymphocytes (hu-PBL-scid mice) (Mosler et al., Nature, 335:256(1988); McCune et al., Science, 241:1632 (1988). Long-term chimerism isachieved in scid mice by injection with human PBL, and antigen-specificsecondary antibody responses are detected in hu-PBL-scid mice challengedin vivo with antigen (Carlsson et al., J. Immunol., 148:1065 (1992);Duchosal et al, Cell Immunol., 139:468 (1992)). This system wasexploited to evaluate the immunosuppressive effects of in vivo anti-gp39administration on the immune responses elicited by human T and B cells.

Experiments, the results of which are contained in FIG. 2B, demonstratedthat blockade of gp39 function by 24-31 inhibited T cell-dependentpolyclonal Ig production by human B cells in vitro. To determine whether24-31 could also inhibit antigen specific B cell antibody production invivo, C.B-17 scid/scid mice injected i.p. with human PBL (hu-PBL-scid)and immunized with tetanus toxoid (TT) were treated with 24-31 or PBS,and the secondary (IgG) anti-TT antibody response was assessed.Immunization of hu-PBL-scid with TT resulted in detectable levels of IgGanti-TT antibody within 14 days post immunization in most animals (Table4). However, treatment with anti-gp39 (24-31; 250 μg/day, twice weekly)completely ablated the secondary anti-TT antibody response in 9/10 miceexamined, demonstrating that in vivo blockade of gp39 function alsoresulted in inhibition of antigen specific humoral responses.

                  TABLE 4                                                         ______________________________________                                        Ablation of the secondary anti-tetanus antibody response following             engraftment of human PBL in C.B-17 scid/scid mice immunized with              tetanus toxoid.* Four-six week old C.B-17-scid/scid mice were injected        i.p. with 20 × 10.sup.6 human PBL and 0.25 ml tetanus toxoid.                      Anti-Tetanus Antibody (O.D. ± SE)§                                     (Frequency of Mice Containing                                      Anti-Tetanus Antibody)                                                      Recipient                                                                            Treat-   days post immunization                                        S train*                                                                             ment¶                                                                        7d      14d    21d     26d                                    ______________________________________                                        C.B-17 PBS      <0.02   2.30 ±                                                                            .224 ± .040                                                                        .137 ± .007                           scid/scid  (0/10) .042 (8/10)** (4/10)                                           (7/10)*                                                                     anti-gp39 .162 <0.02 <0.02 (0/10) <0.02 (0/10)                                 (1/10) (0/10)                                                             ______________________________________                                         ¶Antigp39 2431 or PBS (250 μg/injection) was administered        i.p. twice weekly throughout the entire experiment.                           §The level of human antitetanus toxoid antibody in the serum was         determined weekly by ELISA. All mice with serum levels of human               antitetanus toxoid antibody >0.100 O.D. at a 1.10 dilution were considere     positive. Only positive mice were used in the calculation of the mean .+-     SE values included in the table. The level of human antitetanus toxoid in     sera from preimmune mice not immunized with tetanus toxoid  #was <0.02        O.D. Data are presented as mean ± SE.                                      *Significantly different (p = 0.222) than the antigp39 treated group.         **Significantly different (p < 0.001) than the antigp39 treated group.   

EXAMPLE 4

Anti-gp39 Treatment does not Inhibit the Antigen-Specific T CellProliferative Response of hu-PBL-scid Spleen Cells

To determine whether treatment of hu-PBL-scid mice with anti-gp39altered the responsiveness of antigen-specific T cells in vivo, theproliferative response of spleen cells from hu-PBL-scid mice immunizedwith TT and treated with 24-31 was assessed in vitro. Spleen cells fromcontrol or anti-gp39 treated hu-PBL-scid mice were cultured with TT ormedium alone, and the proliferative response was assessed by ³H-thymidine incorporation after 6 days. Table 5 summarizes the resultsof one such experiment. Hu-PBL-scid mice treated with anti-gp39responded similarly to in vitro stimulation with TT as did hu-PBL-scidmice which were untreated (5/10 vs. 3/10 responding mice). Experimentsusing NOD/LtSz-scid/scid mice as recipients yielded similar results,although anti-TT antibodies were undetectable in these mice (data notshown). These data demonstrate that treatment with anti-gp39 does notresult in deletion or functional inactivation of antigen-specific Tcells in hu-PBL-scid mice and support the contention that inhibition ofTT specific antibody responses by anti-gp39 is due to blockade ofgp39-CD40 interactions and subsequent B cell responses rather than Tcell inactivation.

                  TABLE 5                                                         ______________________________________                                        Anti-gp39 treatment does not alter the anti-tetanus T cell                      proliferative response following engraftment of human PBL in C.B-17-         scid/scid or NOD/LtSz-scid/scid mice immunized with tetanus toxoid.                                      Frequency of Responding                                                        Recipient Strain* Treatment¶                                       Mice§                                        ______________________________________                                        C.B-17 scid/scid                                                                            PBS       3/10                                                     anti-gp39 5/10                                                               NOD/LtSz-scid/scid PBS 5/10                                                    anti-gp39 6/10                                                             ______________________________________                                         *Four-six week old C.B 17scid/scid or NOD/LtSzscid/scid mice were injecte     i.p. with 20 × 10.sup.6 human PBL and 0.25 ml tetanus toxoid.           ¶Antigp39 2431 or PBS (250 μg/injection) was administered        i.p. twice weekly throughout the entire experiment.                           §Spleen cells from mice injected with human PBL and immunized with       tetanus toxoid were cultured at a concentration of 1 × 10.sup.5         cells/ml in the presence of media aione or tetanus toxoid (2.5 or 5.0         μg/ml). Proliferation was assessed by .sup.3 Hthymidine incorporation      after 6d. Stimulation indices were calculated by the following formula"       S.I. = cpm tetanus - cpm medium/cpm medium. S.I. of > than 2.0 was            considered positive.                                                     

EXAMPLE 5

Generation of a gp39 CHO Transfectant Cell Line

Recently, a CHO transfectant that constitutively expresses cell-surfacegp39 was generated to use as a reagent for the humanized anti-gp39 24-31binding studies proposed in this application. The full-length gp39 gene(Hollenbaugh et al, Immunol. Rev., 138:23 (1994)) was amplified bypolymerase chain reaction (PCR) of phytohemagglutinin-activated humanPBL and cloned into IDEC's INPEP4 vector under the transcriptionalcontrol of the cytomegalovirus (CMV) promoter and enhancer elements. ACHO transfectant was established and amplified in 50 nM methotrexate.The transfectant, 50D4, was shown to express cell-surface gp39 by ELISA(data not shown) and FACS analysis (FIG. 3).

EXAMPLE 6

High-Level Expression of Antibodies using a CHO Expression System

IDEC's proprietary N5KG1 expression vector is used in CHO cells forexpression of the humanized anti-gp39 24-31 antibody. This vector isdepicted schematically in FIG. 1. High-level expression of recombinantantibodies is consistently obtained in CHO cells using this vector andsimilar vectors. Using these vectors, a high percentage of G418resistant clones, 5-10%, are found to express significant amounts ofrecombinant proteins (1-10 mg/l of antibody). These are usually singleplasmid copy integrants, and can easily be amplified using methotrexateto obtain 30-100 pg/cell/day of secreted immunoglobulin. Table 6 liststhe antibody levels obtained before and after gene amplification of 3antibodies expressed in CHO cells utilizing this system.

                  TABLE 6                                                         ______________________________________                                        Antibody production levels using IDEC's CHO expression technology                                       after                                                                                    ampiificati after                           before on amplification                                                       amplification in spinner in fermentor                                        Antibody mg/l flask mg/l mg/l                                               ______________________________________                                        Anti-CD4 γ1                                                                       1-2         100-110   950                                             Anti-CD4 γ4 3-4 125-150 N.D.                                            Anti-CD20  5-10 200-300 650                                                 ______________________________________                                    

EXAMPLE 7

Cloning of 24-31 V_(k) and V_(H) DNA Sequences

The anti-gp39 24-31 V_(k) and V_(H) gene segments were cloned andsequenced. Following analyses of their sequences, humanized versions ofthe V region gene segments were designed. The corresponding DNAsequences were synthesized and cloned into a high-level expressionvector containing human constant region genes. A CHO transfectantproducing the humanized 24-31 antibody is then established. To confirmthat the humanized version of the anti-gp39 antibody retains its gp39binding affinity, the relative affinities of the murine and humanizedantibodies were compared in direct binding and competition assays. Inaddition, the ability of the humanized 24-31 to block CD40 binding togp39 and to inhibit T cell-dependent antibody production is evaluated.

1. Cloning of the 24-31 V_(k) and V_(H) gene segments

a. Preparation of cDNA. PolyA⁺ mRNA was prepared from 2×10⁶ cells eachof the 24-31 hybridoma and the NS1 cell line, (Carroll et al, Mol.Immunol., 10:991 (1988)), the fusion partner used in the generation ofthe 24-31 hybridoma, utilizing an Invitrogen Corporation MicroFastTrack™ mRNA isolation kit, according to the manufacturer's protocol.First strand cDNA was synthesized utilizing 50 pmoles oligo-dT and 5units M-MLV reverse transcriptase (Promega) (Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989)) followed by Sephadex G-25 chromatography.

b. PCR amplification Of V_(k) and V_(H) cDNA. 24-31 and NS1 cDNA wereamplified by PCR using a panel of 5' primers specific for V_(k) or V_(H)leader sequences in combination with 3' constant region primers. Thepanel of 5'V_(H) primers are identical to those described by Jones andBendig (Bio/Technol., 9:88 (1991); Errata, Bio/Technol., 9:579 (1991)).The panel of 5'V_(k) primers (Jones et al., (Id.)) were modified toconvert the Sal I cloning site recognition sequences (GTCGAC) into BglII recognition sequences (AGATCT) to facilitate the cloning of theamplified gene segments into IDEC's N5KG1 expression vector (See FIG.1). The 3' V_(k) and V_(H) primers contain a Bsi WI cloning sitesequence at amino acid positions 108-109 (numbering according to Kabatet al., "Sequences of Proteins of Immunological Interest," 5th Ed., NIH(1991)) and a Nhe I cloning site sequence at positions 114-115,respectively, and have the following sequences [SEQ ID NOS.:22-23]:TGCAGCATCCGTACGTTTGATTCCAGCTT(C_(k)) (SEQ ID NO.:22) andGGGGGTGTCGTGCTAGCTG(A/C)(G/A)GAGAC(G/A)GTGA (Cγl)(SEQ ID NO.: 23). Thisprimer panel has been previously used by the Assignee to amplify andclone the C2B8 anti-CD20 antibody (Nishioka et al., J. Immunol.,153:1027 (1994)) and numerous other mouse V_(k) and V_(H) gene segments(data not shown).

In order to determine the correct primer pair for the amplification ofthe 24-31 V_(k) and V_(H) gene segments, the 24-31 cDNA were amplifiedin 23 individual reactions containing one of the 11 5'V_(k) primers incombination with the C_(K) primer or one of the 12 5'V_(H) primers incombination with the Cγ1 primer. For comparison, NS1 cDNA was amplifiedusing the same panel of primers. 1 μl cDNA (1/50 of the cDNA sample) wasamplified in a 100 μl final volume containing 5 units Taq DNA polymerase(Perkin Elmer), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.25 mMeach of dCTP, dGTP, dATP, and TTP, 50 pmoles 3' constant region primer,and 50 pmoles 5' primer. The amplification cycle consisted ofdenaturation for 1 minute at 95° C., annealing for 2 minutes at 50° C.,and extension for 2 minutes at 72° C., repeated 34 times. The amplifiedproducts were analyzed by agarose gel electrophoresis. The 24-31 PCRreactions yielding a unique amplified product for V_(k) and for V_(H)were repeated and the products from duplicate PCR reactions cloned. PCRamplified products are agarose gel-purified (Sambrook et al, MolecularCloning: A Laboratory Manual, 2nd Ed. (1989)) and digested with Bgl IIand Bsi WI (for V_(k)) or Sal I and Nhe I (for V_(H)) The products areligated (Ausabel et al., Current Protocols in Molecular Biology, Vol. 2,Greene Publ. Assoc. (1992)) sequentially into IDEC's vector, N5KG1.

Following transformation of E. coli XL1-blue cells (Stratagene), plasmidDNA was prepared, and the V_(k) and V_(H) sequences obtained from theduplicate constructs (sequencing performed by Scripps Research InstituteCore Facility, La Jolla, Calif.). The sequences of the endogenous lightand heavy chains of the NS1 fusion partner are known (Carroll et al.,Mol. Immunol., 10:991 (1988); Kabat et al., (1991) (Id.)) and were usedto distinguish PCR products resulting from the amplification of the24-31 versus the NS1 fusion partner V regions.

EXAMPLE 8

Synthesis of Gene Segments Encoding Humanized 24-31 V Regions

Humanized versions containing the most preferred humanized 24-31 V_(k)and V_(H) sequences identified in Tables 1 and 2 as humanized V_(L) andV_(H) (1) were synthesized. Specifically, four pairs of overlapping,complementary olionucleotides (oligos) encoding the above-identifiedhumanized V_(k) or V_(H) regions were synthesized (Midland Chemicals)and purified by denaturing polyacrylamide gel electrophoresis (Ausubelet al, Current Protocols in Molecular Biology, Vol. 2, Greene Publ.Assoc. (1992)). Each oligo is approximately 100 bases in length andoverlap by 20 bases the adjacent complementary oligonucleotide. TheV_(k) and V_(H) 5' oligos contain Bgl II and Sal I cloning sites and the3' oligos possess Bsi WI and Nhe I cloning sites, respectively. Eachvariable region gene segment was assembled from the synthetic oligos,diagrammed below, using the following procedure (summarized in Watson etal., Recombinant DNA, 2nd Ed., Scientif. Amer. Books, New York, N.Y.(1992)). Complementary oligo pairs (A+E, B+F, C+G, D+F) were kinasedusing 300 pmoles of each primer and T4 polynucleotide kinase (Promega)according to the manufacturer's protocol. The oligos were annealed byheating to 95° C and slow cooling to room temperature. The annealedoligo pairs were ligated (A/E with B/F and C/G with D/H) utilizing 6units T4 DNA ligase (New England Biolabs). After digestion with theappropriate 5' or 3' cloning site restriction endonuclease, theapproximately 200 base pair DNA fragments were purified byelectroelution following polyacrylamide gel electrophoresis (Sambrook etal, (Id.)). The synthetic gene fragments were then inserted into IDEC'sproprietary high-level expression vector, N5KG1, under thetranscriptional control of the CMV promoter and enhancer elements. Theligation reaction contains the 2 gel-purified fragments (A/E/B/F andC/G/D/H) and N5KG1 at a molar ratio of 100:100:1, respectively. Aftertransformation of XL1-blue cells, plasmid DNA was prepared and thesequences of the synthetic gene segments confirmed. The resultingconstruct, h24-31, encodes the humanized 24-31 V region segments andhuman kappa and gamma 1 constant regions. As indicated, this antibodycontains the humanized variable heavy and humanized variable lightsequences identified in Table 1 and Table 2 as the "(1)" sequences,which are predicted to provide for humanized antibody having optimalgp39 properties. In addition, a construct was generated which containsV_(L) #2 in combination with V_(H) #1 (version 2 of humanized 24-31).Similar constructs utilizing IDEC's proprietary vectors have been usedfor high-level expression of IDEC's anti-CD20 (Reff et al., Blood,83:425 (1994)) and anti-CD4 (Newman et al., Biol. Technology, 10:1455(1992)) antibodies. ##STR1##

EXAMPLE 9

2. Production and Characterization of Humanized 24-31

a. Generation of CHO transfectants producing humanized 24-31 (version 1and version 2).

CHO transfectants expressing humanized 24-31 (version 1 or version 2)were generated by electroporation of 4×10⁶ CHO cells with linearizedh24-31 DNA (version 1 or version 2) followed by selection in G418. Thecell culture supernatants from G418 resistant clones were assayed forimmunoglobulin production by sandwich ELISA employing a goat anti-humankappa to capture the immunoglobulin. Immunoglobulin binding was measuredby incubating with a horse radish peroxidase (HRP)-conjugated goatantibody specific for human IgG, followed by HRP substrate, 0.4 mg/mlO-Phenylene-diamine (OPD) in a citrate buffer (9-34 g/l C₆ H₈ O₇ and14.2 g/l Na₂ HPO₄), pH 5.0, including 0.0175% H₂ O₂. The plate was readin a Molecular DeviCes "Vmax, kinetic microplate reader"spectrophotometer at 490 nm.

EXAMPLE 10

b. Characterization of humanized 24-31 (version 1).

The humanized anti-gp39 24-31 antibody is evaluated initially for directbinding to cell surface gp39 expressed on 50D4, the gp39 CHOtransfectant described in Example 5. Supernatants from theG418-resistant h24-31 CHO transfectants that produce immunoglobulin aretested for binding to 50D4 cells and, as negative control, to CHO cells.In this assay 50D4, 1×10⁵ /well, are bound to the bottom of 96 well,poly-L-lysine coated polystyrene plates. The cells are fixed in 0.5%glutaraldehyde in phosphate buffered saline (PBS) for 15 minutes. Platescoated with CHO cells are generated similarly. The cell culturesupernatants are added and antibody binding measured using aHRP-conjugated goat anti-human IgG, as described above.

Two assays are used to determine if the humanized 24-31 antibody retainsits affinity to gp39 relative to the original murine 24-31 antibody, (i)half-maximal binding concentration and (ii) a competition assay using50D4 cells. For this purpose the antibodies will be purified on proteinA and the concentration of each antibody determined by ELISA by acomparison to isotype matched controls. Half-maximal binding (i) aredetermined by incubating humanized 24-31 with 50D4 cells at variousconcentrations from 2 μg/ml to 0.1 ng/ml. The concentration resulting ina half-maximal OD 490 reading, as described above, is compared with thehalf-maximal binding of murine 24-31. In the competition assay (ii) thehumanized 24-31 antibody and the murine 24-31 antibody are mixed invarious molar ratios ranging from 100:1 to 1:100, and their ability tocompete for binding to 50D4 cells measured. Two sets are run, one wherethe binding of the humanized antibody will be measured usinggoat-anti-human IgG (anti-mouse IgG depleted)-HRP and one where thebinding of murine antibody is measured using goat-anti-mouse IgG(anti-human IgG depleted)-HRP. Binding curves, one for the murine andone for the humanized antibody, based on molar ratios, are generated andtheir relative affinities calculated. These assays will confirm theanti-gp39 binding properties of the subject humanized antibodies derivedfrom 24-31.

EXAMPLE 11

Blocking of CD40-Ig binding to gp39 by humanized 24-31.

After establishing that humanized anti-gp39 binds to gp39, an assay iseffected to confirm that the humanized anti-gp39 retains its ability toblock the binding of the ligand to its receptor. For this purpose,activated human peripheral blood T cells, or the gp39-transfected CHOcells, 50D4, are pretreated with graded concentrations of murine 24-31or with humanized 24-31 for 15 minutes at 4° C.

Following this preincubation, CD40-Ig-biotin is added and the bindingdetermined by flow cytometry using PE-avidin. Concentrations of mabs toachieve a 50% reduction in CD40-Ig binding are determined.

EXAMPLE 12

Blocking of B cell proliferation and differentiation by humanized 24-31.

To confirm that humanized 24-31 blocks gp39 function, B cells arecultured with a soluble fusion protein of gp39 (gp39-CD8) in thepresence or absence of a range of doses of murine 24-31 or humanized24-31. B cell proliferative response is assessed by ³ H-thymidineincorporation as shown in FIG. 2A.

T cell dependent B cell differentiation (Ig production) is blocked bymAbs to gp39. To confirm that the subject humanized 24-31 antibodies areeffective in blocking the function of native gp39 expressed on thesurface of activated human T cells, the ability of the subject humanized24-31 antibodies inhibit T cell-induced B cell differentiation isassessed. B cells are co-cultured with anti-CD3 activated T cells in thepresence or absence of humanized 24-31 and murine 24-31. Polyclonal IgM,IgG, and IgA production is assessed after 12 days (see FIG. 2B).

These results will confirm that humanized anti-gp39 can block CD40binding and interfere with T-cell-dependent B cell activation via CD40.

EXAMPLE 13

Binding Capacity

This experiment was effected to determine the reactivity of the murine,chimeric, and humanized (version 1) 24-31 antibodies to the gp39 antigenrelative to the concentration of antibody.

Protocol

Plate Preparation

1. Add 50 of poly-1-lysine to each well on the 96 well plate. Incubatefor 30 minutes at room temperature. Flick plates to removepoly-1-lysine.

2. Wash mgp39-CHO cells (Chinese hamster ovary cells expressing cellsurface, membrane gp39) 3 times with HBSS by centrifuging at 1500 rpmfor 5 minutes. Resuspend cells in HBSS to 2×10⁶ cells ml.

3. Add 50 μl of cell suspension to each well and centrifuge plates at2000 rpm for 5 minutes.

4. Add 50 μl/well of ice cold 0.5% glutaraldehyde and incubate for 15minutes at room temperature.

5. Flick plate and blot to remove excess glutaraldehyde. Add 150 μl/wellof 100 mM glycine with 0.1%. BSA and incubate for 30 minutes at roomtemperature. Plates can be used immediately or frozen at -20° C. forfuture use.

Binding Assay

1. Thaw plate and remove glycine buffer.

2. Serially dilute, 1:2, the test antibodies in dilution buffer startingat 1 μg/ml. Transfer 50 μg/well of each dilution in duplicate. Incubate2 hours at room temperature.

3. Wash plate 10 times in flowing tap water.

4. Add 50 μl/well of 1:2000 dilution of goat anti-human IgG HRP or goatanti-mouse IgG HRP. Incubate 1 hour at room temperature.

5. Wash plate 10 times in flowing tap water.

6. Add 50 μl/well of ABTS substrate and develop plate for 20-30 minutes.Read the plate at wavelength 405 mn with a background wavelength of 490run.

7. Plot graph of absorbance vs antibody concentration.

Results and Conclusions

The binding capacities for the three anti-gp39 antibodies (murine,chimeric and humanized version 1 of 24-31) relative to the concentrationof the antibodies, were essentially superimposable (see FIG. 9). This isa good indication that these antibodies have similar binding capacitiesfor human gp39, indicating that the humanized antibody has retained thegp39 binding affinity of murine 24-31.

EXAMPLE 14

Competition Between Biotin Labeled Murine 24-31 and Chimeric andHumanized Version 1 24-31 The ability of the chimeric and humanized(version 1) 24-31 antibodies to compete with the murine 24-31 forbinding to mgp39-CHO cells basis was evaluated. The ability of thehumanized 24-31 to compete with the murine 24-31 for binding tomgp39-CHO was used to evaluate whether in the humanized antibody theexchanges of the murine framework residues with their human counterpartsresulted in a significant loss (≧3× decrease) of affinity.

Protocol

Plate Preparation

1. Add 50 of poly-1-lysine to each well on the 96 well plate. Incubatefor 30 minutes at room temperature. Flick plates to removepoly-1-lysine.

2. Wash mgp39-CHO cells 3 times with HBSS by centrifuging at 1500 rpmfor 5 minutes. Resuspend cells in HBSS to 2×10⁶ cells/ml.

3. Add 50 μl of cell suspension to each well and centrifuge plates at2000 rpm for 5 minutes.

4. Add 50 μl/well of ice cold 0.5% glutaraldehyde and incubate for 15minutes at room temperature.

5. Flick plate and blot to remove excess glutaraldehyde.

Add 150 μl/well of 100 mM glycine with 0.1% BSA and incubate for 30minutes at room temperature. Plates can be used immediately or frozen at-20° C. for future use.

Competition Assay

1. Thaw plate and remove glycine buffer.

2. Dilute mouse anti-gp39 biotin to 200 ng/ml in PBS with 1% BSA.

3. Serially dilute test antibodies (mouse, chimeric, and humanized24-31) 1:2 starting at 10 μg/ml in dilution buffer.

4. Transfer 50 μl of diluted test antibodies and mouse anti-gp39 biotininto each well in duplicate. Several wells should contain 50 μl dilutionbuffer with the mouse anti-gp39 biotin as a maximal control group.Incubate 2 hours at room temperature.

5. Wash plates 10 times in flowing tap water.

6. Add 50 μl/well of 1:2000 dilution of streptavidin HRP and incubate 1hour at room temperature.

7. Wash plates 10 times in flowing tap water.

8. Add 50 μl/well of ABTS substrate and develop plate for 20-30 minutes.Read the plate at wavelength 405 nm with a background wavelength of 490nm.

9. Percent inhibition is calculated using the average of the controlwells.

Results and Conclusions

All three antibodies competed equally well with the biotin labeled 24-31(see FIG. 10). The competition profiles are essentially superimposableat all concentrations, within the limitations of the assay. Thisdemonstrates that the tested humanized antibody (version 1) retains itsgp39 binding affinity.

EXAMPLE 15 Modulation of T Cell Dependent B Cell Differentiation

To confirm that the humanized 24-31 retains the in vitro functionalactivity of murine 24-31, the humanized 24-31 was compared to the murine24-31 in a "Lipsky" assay. Donor peripheral blood mononuclear cells wereseparated into two fractions, a T and a B cell fraction. The T cellswere first treated with mitomycin C, to prevent mitosis, and thenactivated with an anti-CD3 antibody. The B cells were added, togetherwith either the murine or humanized (version 1) 24-31 antibodies. Apositive control without antibody, and a negative control without Bcells were included in the experiment. After a 10 day incubation, thesupernatants were tested for the presence of human IgM.

Protocol

1. Coat a 96 well plate with 50 μl/well of sterile 4 μg/ml anti-CD3antibody (diluted in 50 mM Tris, pH 9) for 2 hours at 37° C.

2. Selectively purify T and B cells from a buffy coat using Lympho-Kwikreagents. Activate the T cells with 50 μg/ml mitomycin C per 5×10⁶ cellsfor 30 minutes at 37° C.

3. Wash plate wells several times with sterile HBSS or media to removenonadherent antibody.

4. Add 1×10⁵ purified T cells (2×10⁶ /ml) to each well.

5. Add 5×10⁵ purified B cells (5×10⁶ /ml) to each well. Add 50 μlanti-gp39 antibody (10-0.1 μg/ml) to each well in quadruplicate. Controlwells should include: a) 0 antibody, b) 0 antibody, no T cells, and c) 0antibody, no B cells.

6. Incubate plate at 37° C./5% CO₂ for 12 days.

7. Access cell growth after 7 days using 3H thymidine or any otheracceptable method on duplicate wells.

8. After 12 days, collect supernatants from duplicate wells and performELISA assays to determine Ig production (IgM).

Results and Conclusions

The results show that the production of human IgM is inhibited 50% bythe humanized 24-31 at a concentration below 0.01 μg/ml, similar to theinhibition level obtained with the murine 24-31 (see FIG. 11). Thehumanized antibody retained its ability to inhibit T cell dependent Bcell differentiation (IgM production) in this experiment.

EXAMPLE 16 Evaluation of Humanized 24-31, Version 2

This experiment was conducted to determine whether humanized 24-31version 2, as compared to version 1, has a similar gp39 binding capacityin a direct binding assay.

Protocol

Same as in Example 13 above.

Results and Conclusions

The results show that the binding capacity of the two 24-31 versions areessentially superimposable (see FIG. 12). This indicates that the twoversions have comparable binding activity to gp39.

EXAMPLE 17

This experiment was conducted to measure the Kd of 24-31, and twohumanized versions, 1 and 2.

Protocol

A predetermined amount of each of the three antibodies (murine, version1 or version 2 24-31) was labeled with ¹²⁵ I using IODO-BEADS® (Pierce).Antibody bound-¹²⁵ I was separated from free ¹²⁵ I by size separation ona Sephadex-G25/DEAE/Amberlite column.

Direct binding of the ¹²⁵ I-labeled antibody to murine gp39-CHO cellswas tested in a dilution series, in order to determine both counts/μgand the appropriate working concentration (˜half-maximal bindingconcentration).

¹²⁵ I-labeled antibody was mixed and incubated with non-labeled antibodyin a dilution series. Based on the total amount of bound antibody andthe amount of free antibody, a Scatchard plot was generated from a boundvs. bound-free graph. The total antibody concentration was based on astandard size of 75 kD for one active site.

The Kd was calculated by generating a "best fit" line. The inverse ofthe slope of the curve is the Kd. The correlation coefficient, r², wasalso computed.

Results

The Scatchard plots w ere analyzed. The Kd's from this analysis are:Version 2, Kd=14 nM; murine 24-31, Kd=8.51 nM; version 1, Kd=5.6. Theresults are depicted in FIGS. 13, 14 and 15, respectively. These resultsprovide further evidence that the subject humanized antibodies bind thegp39 antigen similarly to 24-31.

Use

The humanized anti-gp39 antibodies of the present invention havepotential in treating any disease condition wherein gp39 modulationand/or inhibition of the gp39-CD40 interaction is therapeuticallybeneficial. Moreover, the subject humanized anti-gp39 antibodies may beused in treatment of diseases wherein suppression of antibody responsesto antigens are desirable. Such conditions include both autoimmune andnon-autoimmune disorders.

The ability of anti-gp39 antibodies to prevent CD40 signaling in B cellsis functionally translated into marked inhibition of T cell-dependentantibody responses in vivo. Therefore, autoimmune diseases which aremediated by autoantibody production would be expected to benefit fromanti-gp39 antibody therapy. Such diseases include systemic lupuserythematosus, idiopathic thrombocytopenic purpura, myasthenia gravisand a subpopulation of diabetic patients with anti-insulin andanti-insulin receptor antibodies. In addition, CD40 signaling in B cellsand dendritic cells is essential for upregulation of co-signalingreceptors such as B7.1 and B7.2 molecules. Blocking of this CD40signaling by is anti-gp39 antibodies interferes with antigenpresentation to T cells, resulting in inhibition of T cell activationand T cell-mediated responses. The therapeutic efficacy of anti-gp39antibodies in disease models such as CIA, EAE, NOD mice, GVHD and graftrejection further confirms the antibody's inhibitory effect on Tcell-mediated responses. Based on this mechanism of action supported bythe efficacy in animal models, the therapeutic potential of the subjecthumanized anti-gp39 antibodies extend to such diseases as RA, MS,diabetes, psoriasis, GVHD and graft rejection.

Specific conditions which are potentially treatable by administration ofthe subject humanized antibodies include the following:

Allergic bronchopulmonary aspergillosis; Autoimmune hemolytic anemia;Acanthosis nigricans; Allergic contact dermatitis; Addison's disease;Atopic dermatitis; Alopecia areata; Alopecia universalis; Amyloidosis;Anaphylactoid purpura; Anaphylactoid reaction; Aplastic anemia;Angioedema, hereditary; Angioedema, idiopathic; Ankylosing spondylitis;Arteritis, cranial; Arteritis, giant cell; Arteritis, Takayasu's;Arteritis, temporal; Asthma; Ataxia-telangiectasia; Autoimmuneoophoritis; Autoimmune orchitis; Autoimmune polyendocrine failure;Behcet's disease; Berger's disease; Buerger's disease; Bullouspemphigus; Candidiasis, chronic mucocutaneous; Caplan's syndrome;Post-myocardial infarction syndrome; Post-pericardiotomy syndrome;Carditis; Celiac sprue; Chagas's disease; Chediak-Higashi syndrome;Churg-Strauss disease; Cogan's syndrome; Cold agglutinin disease; CRESTsyndrome; Crohn's disease; Cryoglobulinemia; Cryptogenic fibrosingalveolitis; Dermatitis herpetifomis; Dermatomyositis; Diabetes mellitus;Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupuserythematosus; Eosinophilic fasciitis; Episcleritis; Drythema elevatumdiutinum; Erythcma marginatum; Erythema multiforme; Erythema nodosum;Familial Mediterranean fever; Felty's syndrome; Fibrosis pulmonary;Glomerulonephritis, anaphylactoid; Glomerulonephritis, autoimmune;streptococcphritis, post-streptococcal; Glomerulonephritis,post-transplantation; Glomerulopathy, membranous; Goodpasture'ssyndrome; Graft-vs.-host disease; Granulocytopenia, immune-mediated;Granuloma annulare; Granulomatosis, allergic; Granulomatous myositis;Grave's disease; Hashimoto's thyroiditis; Hemolytic disease of thenewborn; Hemochromatosis, idiopathic; Henoch-Schoenlein purpura;Hepatitis, chronic active and chronic progressive; Histiocytosis X;Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura; Job'ssyndrome; Juvenile dermatomyositis; Juvenile rheumatoid arthritis(Juvenile chronic arthritis); Kawasaki's disease; Keratitis;Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl syndrome;Leprosy, lepromatous; Loeffler's syndrome; Lyell's syndrome; Lymedisease; Lymphomatoid granulomatosis; Mastocytosis, systemic; Mixedconnective tissue disease; Mononeuritis multiplex; Muckle-Wellssyndrome; Mucocutaneous lymph node syndrome; Mucocutaneous lymph nodesyndrome; Multicentric reticulohistiocytosis; Multiple sclerosis;Myasthenia gravis; Mycosis fungoides; Necrotizing vasculitis, systemic;Nephrotic syndrome; Overlap syndrome; Panniculitis; Paroxysmal coldhemoglobinuria; Paroxysmal nocturnal hemoglobinuria; Pemphigoid;Pemphigus; Pemphigus erythematosus; Pemphigus foliaceus; Pemphigusvulgaris; Pigeon breeder's disease; Pneumonitis, hypersensitivity;Polyarteritis nodosa; Polymyalgia rheumatica; Polymyositis;Polyneuritis, idiopathic; Portuguese familial polyneuropathics;Pre-eclampsia/eclampsia; Primary biliary cirrhosis; Progressive systemicsclerosis (Scleroderma); Psoriasis; Psoriatic arthritis; Pulmonaryalveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon/syndrome;Reidel's thyroiditis; Reiter's syndrome, Relapsing polychrondritis;Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis;Sclerosing cholangitis; Serum sickness; Sezary syndrome; Sjogren'ssyndrome; Stevens-Johnson syndrome; Still's disease; Subacute sclerosingpanencephalitis; Sympathetic ophthalmia; Systemic lupus erythematosus;Transplant rejection; Ulcerative colitis; Undifferentiated connectivetissue disease; Urticaria, chronic; Urticaria, cold; Uveitis; Vitiligo;Weber-Christian disease; Wegener's granulomatosis; Wiskott-Aldrichsyndrome.

Of these, the preferred indications treatable or presentable byadministration of anti-gp39 antibodies include autoimmune hemolyticanemia; aplastic anemia; arteritis, temporal; diabetes mellitus; Felty'ssyndrome; Goodpasture's syndrome; graft-vs-host disease; idiopathicthrombocytopenia pupura; myasthenia gravis; multiple sclerosis;polyarteritis nodosa; psoriasis; psoriatic arthritis; rheumatoidarthritis; systemic lupus erythematosus; asthma; allergic conditions;and transplant rejection.

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. Because of the efficacy of thepresently claimed antibodies and their tolerance by humans it ispossible to administer these antibodies repetitively in order to combatvarious diseases or disease states within a human.

The subject anti-gp39 humanized antibodies (or fragments thereof) ofthis invention are also useful for inducing immunomodulation, e.g.,inducing suppression of a human's or animal's immune system. Thisinvention therefore relates to a method of prophylactically ortherapeutically inducing immunomodulation in a human or other animal inneed thereof by administering an effective, non-toxic amount of such anantibody of this invention to such human or other animal.

The fact that the antibodies of this invention have utility in inducingimmunosuppression means that they are useful in the treatment orprevention of resistance to or rejection of transplanted organs ortissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.);the treatment or prevention of autoimmune, inflammatory, proliferativeand hyperproliferative diseases, and of cutaneous manifestations ofimmunologically mediated diseases (e.g., rheumatoid arthritis, lupuserythematosus, systemic lupus erythematosus, Hashimotos thyroiditis,multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis,nephrotic syndrome, psoriasis, atopical dermatitis, contact dermatitisand further eczematous dermatitides, seborrheic dermatitis, Lichenplanus, Pemplugus, bullous pemphicjus, Epidermolysis bullosa, urticaria,angioedemas, vasculitides, erythema, cutaneous eosinophilias, Alopeciaareata, etc.); the treatment of reversible obstructive airways disease,intestinal inflammations and allergies (e.g., Coeliac disease,proctitis, eosinophilia gastroenteritis, mastocytosis, Crohn's diseaseand ulcerative colitis) and food-related allergies (e.g., migraine,rhinitis and eczema). Also, the subject antibodies have potentialutility for treatment of non-autoimmune conditions whereinimmunomodulation is desirable, e.g., graft-versus-host disease (GVHD),transplant rejection, asthma, leukemia, lymphoma, among others.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody would be forthe purpose of inducing immunosuppression. Generally, however, aneffective dosage will be in the range of about 0.05 to 100 milligramsper kilogram body weight per day.

The antibodies of the invention may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce such effect to a therapeutic orprophylactic degree. Such antibodies of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody (or fragment thereof) of theinvention may be oral, parenteral, by inhalation or topical. The termparenteral as used herein includes intravenous, intramuscular,subcutaneous, rectal, vaginal or intraperitoneal administration. Thesubcutaneous and intramuscular forms of parenteral administration aregenerally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression will generally be in the range of about 0.05 to 100,but preferably about 0.5 to 10, milligrams per kilogram body weight perday.

The antibody of the invention may also be administered by inhalation. By"inhalation" is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 10 to 100milligrams.

The antibody of the invention may also be administered topically. Bytopical administration is meant non-systemic administration and includesthe application of an antibody (or fragment thereof) compound of theinvention externally to the epidermis, to the buccal cavity andinstillation of such an antibody into the ear, eye and nose, and whereit does not significantly enter the blood stream. By systemicadministration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody required fortherapeutic or prophylactic effect will, of course, vary with theantibody chosen, the nature and severity of the condition being treatedand the animal undergoing treatment, and is ultimately at the discretionof the physician. A suitable topical dose of an antibody of theinvention will generally be within the range of about 1 to 100milligrams per kilogram body weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carrier(s) therefor andoptionally any other therapeutic ingredients(s). The carrier(s) must be"acceptable" in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at90°-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogols. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an antibody or fragmentthereof of the invention will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular animal being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of an antibody or fragment thereof of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following are, therefore, to be construed asmerely illustrative examples and not a limitation of the scope of thepresent invention in any way.

Capsule Composition

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg. of an antibody or fragment thereof of the invention, in powderedform, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesiumstearate.

Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5 k by weight ofan antibody or fragment thereof of the invention in 10 k by volumepropylene glycol and water. The solution is sterilized by filtration.

Ointment Composition

Antibody or fragment thereof of the invention 1.0 g.

White soft paraffin to 100.0 g.

The antibody or fragment thereof of the invention is dispersed in asmall volume of the vehicle to produce a smooth, homogeneous product.Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition

Antibody or fragment thereof of the invention 1.0 g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60° C. Asolution of methyl hydroxybenzoate is added and homogenization isachieved using high speed stirring. The temperature is then allowed tofall to SOOC. The antibody or fragment thereof of the invention is thenadded and dispersed throughout, and the composition is allowed to coolwith slow speed stirring.

Topical Lotion Composition

Antibody or fragment thereof of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g. Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g. Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified Water B.P. to 100.00 ml. (B.P.=British Pharmacopeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the antibody or fragment thereof of the invention is addedas a suspension in the remaining water. The whole suspension is stirreduntil homogenized.

Eve Drop Composition

Antibody or fragment thereof of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B.P. to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purifiedwater at 75° C. and the resulting solution is allowed to cool. Theantibody or fragment thereof of the invention is then added, and thesolution is sterilized by filtration through a membrane filter (0.022 μmpore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: mix 10 mg. of anantibody or fragment thereof of the invention with 0.2-0.5 k of alubricating agent, such as polysorbate 85 or oleic acid, and dispersesuch mixture in a propellant, such as freon, preferably in a combinationof (1,2 dichlorotetrafluoroethane) and difluorochloromethane and putinto an appropriate aerosol container adapted for either intranasal ororal inhalation administration. Composition for Administration byInhalation For an aerosol container with a capacity of 15-20 ml:dissolve 10 mg. of an antibody or fragment thereof of the invention inethanol (6-8 ml.), add 0.1-0.2 k of a lubricating agent, such aspolysorbate 85 or oleic acid; and disperse such in a propellant, such asfreon, preferably in combination of (1-2 dichlorotetrafluoroethane) anddifluorochloromethane, and put into an appropriate aerosol containeradapted for either intranasal or oral inhalation administration.

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The compositions for parenteraladministration will commonly comprise a solution of an antibody orfragment thereof of the invention or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., water, buffered water, 0.4 k saline,0.3% glycine, and the like. These solutions are sterile and generallyfree of particulate matter. These solutions may be sterilized byconventional, well-known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, etc. The concentration of the antibody or fragment thereof ofthe invention in such pharmaceutical formulation can vary widely, i.e.,from less than about 0.5 k, usually at or at least about 1% to as muchas 15 or 20% by weight, and will be selected primarily based on fluidvolumes, viscosities, etc., according to the particular mode ofadministration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, and50 mg. of an antibody or fragment thereof of the invention. Similarly, apharmaceutical composition of the invention for intravenous infusioncould be made up to contain 250 ml. of sterile Ringer's solution, and150 mg. of an antibody or fragment thereof of the invention. Actualmethods for preparing parenterally administrable compositions arewell-known or will be apparent to those skilled in the art, and aredescribed in more detail in, for example, Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa., herebyincorporated by reference herein.

The antibodies (or fragments thereof) of the invention can belyophilized for storage and reconstituted in a suitable carrier prior touse. This technique has been shown to be effective with conventionalimmune globulins and art-known lyophilization and reconstitutiontechniques can be employed.

Depending on the intended result, the pharmaceutical composition of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already suffering from a disease, in an amount sufficient tocure or at least partially arrest the disease and its complications. Inprophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. In any event, the pharmaceutical composition of theinvention should provide a quantity of the altered antibodies (orfragments thereof) of the invention sufficient to effectively treat thepatient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody. See, e.g., Saragovi et al., Science, 253:792-795 (1991).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without diverting fromthe scope of the invention. Accordingly, the invention is not limited bythe appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 28                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -      Asp Ile Val Met Thr Gln Ser Pro - # Ser Phe Leu Ser Ala Ser       Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Thr Ile Thr Cys Lys - # Ala Ser Gln Asn Val Ile       Thr Ala                                                                                          20 - #                 25 - #                 30             - -      Val Ala Trp Tyr Gln Gln Lys Pro - # Gly Lys Ser Pro Lys Leu        Leu Ile                                                                                      35     - #             40     - #             45                  - -      Tyr Ser Ala Ser Asn Arg Tyr Thr - # Gly Val Pro Asp Arg Phe       Ser Gly                                                                                  50         - #         55         - #         60                      - -      Ser Gly Ser Gly Thr Asp Phe Thr - # Leu Thr Ile Ser Ser Leu       Gln Pro                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Glu Asp Phe Ala Asp Tyr Phe Cys - # Gln Gln Tyr Asn Ser Tyr        Pro Tyr                                                                                           - #   85               - #   90               - #         95                                                                               - -      Thr Phe Gly Gly Gly Thr Lys Leu - # Glu Ile Lys                                      100 - #                105                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -      Asp Ile Val Met Thr Gln Ser Pro - # Asp Ser Leu Ala Val Ser       Leu Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Arg Ala Thr Ile Asn Cys Lys - # Ala Ser Gln Asn Val Ile       Thr Ala                                                                                          20 - #                 25 - #                 30             - -      Val Ala Trp Tyr Gln Gln Lys Pro - # Gly Gln Ser Pro Lys Leu        Leu Ile                                                                                      35     - #             40     - #             45                  - -      Tyr Ser Ala Ser Asn Arg Tyr Thr - # Gly Val Pro Asp Arg Phe       Ser Gly                                                                                  50         - #         55         - #         60                      - -      Ser Gly Ser Gly Thr Asp Phe Thr - # Leu Thr Ile Ser Ser Leu       Gln Ala                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Glu Asp Val Ala Asp Tyr Phe Cys - # Gln Gln Tyr Asn Ser Tyr        Pro Tyr                                                                                           - #   85               - #   90               - #         95                                                                               - -      Thr Phe Gly Gly Gly Thr Lys Leu - # Glu Ile Lys                                      100 - #                105                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -      Asp Ile Val Met Thr Gln Ser Pro - # Ser Phe Met Ser Thr Ser       Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Thr Ile Thr Cys Lys - # Ala Ser Gln Asn Val Ile       Thr Ala                                                                                          20 - #                 25 - #                 30             - -      Val Ala Trp Tyr Gln Gln Lys Pro - # Gly Lys Ser Pro Lys Leu        Leu Ile                                                                                      35     - #             40     - #             45                  - -      Tyr Ser Ala Ser Asn Arg Tyr Thr - # Gly Val Pro Asp Arg Phe       Ser Gly                                                                                  50         - #         55         - #         60                      - -      Ser Gly Ser Gly Thr Asp Phe Thr - # Leu Thr Ile Ser Ser Met       Gln Pro                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Glu Asp Phe Ala Asp Tyr Phe Cys - # Gln Gln Tyr Asn Ser Tyr        Pro Tyr                                                                                           - #   85               - #   90               - #         95                                                                               - -      Thr Phe Gly Gly Gly Thr Lys Leu - # Glu Ile Lys                                      100 - #                105                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -      Asp Ile Val Met Thr Gln Ser Pro - # Asp Ser Met Ala Thr Ser       Leu Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Arg Val Thr Ile Asn Cys Lys - # Ala Ser Gln Asn Val Ile       Thr Ala                                                                                          20 - #                 25 - #                 30             - -      Val Ala Trp Tyr Gln Gln Lys Pro - # Gly Gln Ser Pro Lys Leu        Leu Ile                                                                                      35     - #             40     - #             45                  - -      Tyr Ser Ala Ser Asn Arg Tyr Thr - # Gly Val Pro Asp Arg Phe       Ser Gly                                                                                  50         - #         55         - #         60                      - -      Ser Gly Ser Gly Thr Asp Phe Thr - # Leu Thr Ile Ser Ser Met       Gln Ala                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Glu Asp Val Ala Asp Tyr Phe Cys - # Gln Gln Tyr Asn Ser Tyr        Pro Tyr                                                                                           - #   85               - #   90               - #         95                                                                               - -      Thr Phe Gly Gly Gly Thr Lys Leu - # Glu Ile Lys                                      100 - #                105                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Asp Ser Ile Thr       Asn Gly                                                                                          20 - #                 25 - #                 30             - -      Phe Trp Ile Trp Ile Arg Lys Pro - # Pro Gly Asn Lys Leu Glu        Tyr Met                                                                                      35     - #             40     - #             45                  - -      Gly Tyr Ile Ser Tyr Ser Gly Ser - # Thr Tyr Tyr Asn Pro Ser       Leu Lys                                                                                  50         - #         55         - #         60                      - -      Ser Arg Ile Ser Ile Ser Arg Asp - # Thr Ser Lys Asn Gln Phe       Ser Leu                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Lys Leu Ser Ser Val Thr Ala Ala - # Asp Thr Gly Val Tyr Tyr        Cys Ala                                                                                           - #   85               - #   90               - #         95                                                                               - -      Cys Arg Ser Tyr Gly Arg Thr Pro - # Tyr Tyr Phe Asp Phe Trp       Gly Gln                                                                                          100 - #                105 - #                110            - -      Gly Thr Thr Leu Thr Val Ser Ser                                                  115     - #            120                                        - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Asp Ser Ile Thr       Asn Gly                                                                                          20 - #                 25 - #                 30             - -      Phe Trp Ile Trp Ile Arg Lys His - # Pro Gly Asn Lys Leu Glu        Tyr Met                                                                                      35     - #             40     - #             45                  - -      Gly Tyr Ile Ser Tyr Ser Gly Ser - # Thr Tyr Tyr Asn Pro Ser       Leu Lys                                                                                  50         - #         55         - #         60                      - -      Ser Arg Ile Ser Ile Ser Arg Asp - # Thr Ser Lys Asn Gln Phe       Ser Leu                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Lys Leu Ser Ser Val Thr Ala Ala - # Asp Thr Gly Val Tyr Tyr        Cys Ala                                                                                           - #   85               - #   90               - #         95                                                                               - -      Cys Arg Ser Tyr Gly Arg Thr Pro - # Tyr Tyr Phe Asp Phe Trp       Gly Gln                                                                                          100 - #                105 - #                110            - -      Gly Thr Thr Leu Thr Val Ser Ser                                                  115     - #            120                                        - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Ala Val - # Ser Gly Asp Ser Ile Thr       Asn Gly                                                                                          20 - #                 25 - #                 30             - -      Phe Trp Ile Trp Ile Arg Lys His - # Pro Gly Asn Lys Leu Glu        Tyr Met                                                                                      35     - #             40     - #             45                  - -      Gly Tyr Ile Ser Tyr Ser Gly Ser - # Thr Tyr Tyr Asn Pro Ser       Leu Lys                                                                                  50         - #         55         - #         60                      - -      Ser Arg Ile Ser Ile Ser Arg Asp - # Thr Ser Asn Asn Gln Phe       Ser Leu                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Asn Leu Asn Ser Val Thr Arg Ala - # Asp Thr Gly Val Tyr Tyr        Cys Ala                                                                                           - #   85               - #   90               - #         95                                                                               - -      Cys Arg Ser Tyr Gly Arg Thr Pro - # Tyr Tyr Phe Asp Phe Trp       Gly Gln                                                                                          100 - #                105 - #                110            - -      Gly Thr Thr Leu Thr Val Ser Ser                                                  115     - #            120                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Ala Val - # Tyr Gly Asp Ser Ile Thr       Asn Gly                                                                                          20 - #                 25 - #                 30             - -      Phe Trp Ile Trp Ile Arg Lys Pro - # Pro Gly Asn Lys Leu Glu        Tyr Met                                                                                      35     - #             40     - #             45                  - -      Gly Tyr Ile Ser Tyr Ser Gly Ser - # Thr Tyr Tyr Asn Pro Ser       Leu Lys                                                                                  50         - #         55         - #         60                      - -      Ser Arg Ile Ser Ile Ser Arg Asp - # Thr Ser Lys Asn Gln Phe       Tyr Leu                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Lys Leu Ser Ser Val Thr Ala Ala - # Asp Thr Gly Val Tyr Tyr        Cys Ala                                                                                           - #   85               - #   90               - #         95                                                                               - -      Cys Arg Ser Tyr Gly Arg Thr Pro - # Tyr Tyr Phe Asp Phe Trp       Gly Gln                                                                                          100 - #                105 - #                110            - -      Gly Thr Thr Leu Thr Val Ser Ser                                                  115     - #            120                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -      Asp Ile Gln Met Thr Gln Ser Pro - # Ser Phe Leu Ser Ala Ser        Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Thr Ile Thr Cys Trp - # Tyr Gln Gln Lys Pro Gly       Lys Ala                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Leu Leu Ile Tyr Gly Val - # Pro Ser Arg Phe Ser Gly        Ser Gly                                                                                      35     - #             40     - #             45                  - -      Ser Gly Thr Asp Phe Thr Leu Thr - # Ile Ser Ser Leu Gln Pro       Glu Asp                                                                                  50         - #         55         - #         60                      - -      Phe Ala Thr Tyr Tyr Cys                                                  65             - #     70                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -      Asp Ile Gln Met Thr Gln Ser Pro - # Ser Thr Leu Ser Ala Ser       Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Thr Ile Thr Cys Trp - # Tyr Gln Gln Lys Pro Gly       Glu Ala                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Val Leu Ile Tyr Gly Val - # Pro Ser Arg Phe Ser Gly        Ser Gly                                                                                      35     - #             40     - #             45                  - -      Ser Gly Thr Glu Phe Thr Leu Thr - # Ile Ser Ser Leu Gln Ser       Asp Asp                                                                                  50         - #         55         - #         60                      - -      Phe Ala Thr Tyr Tyr Cys Phe Gly - # Gln Gly Thr Lys Leu Glu       Ile Lys                                                                              65             - #     70             - #     75             - #         80                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - -      Asp Ile Val Met Thr Gln Ser Gln - # Lys Phe Met Ser Thr Ser        Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Ser Ile Thr Cys Trp - # Tyr Gln Gln Lys Pro Gly       Gln Ser                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Ile Leu Leu Ile Tyr Gly - # Val Pro Asp Arg Phe Ser        Gly Ser                                                                                      35     - #             40     - #             45                  - -      Gly Ser Gly Thr Asp Phe Thr Leu - # Thr Ile Ser Asn Met Gln       Ser Glu                                                                                  50         - #         55         - #         60                      - -      Asp Leu Ala Asp Tyr Phe Cys Phe - # Gly Gly Gly Thr Lys Leu       Glu Ile                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Lys                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - -      Asp Ile Val Met Thr Gln Ser Pro - # Ser Phe Leu Ser Ala Ser        Val Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Arg Val Thr Ile Thr Cys Trp - # Tyr Gln Gln Lys Pro Gly       Lys Ser                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Leu Leu Ile Tyr Gly Val - # Pro Asp Arg Phe Ser Gly        Ser Gly                                                                                      35     - #             40     - #             45                  - -      Ser Gly Thr Asp Phe Thr Leu Thr - # Ile Ser Ser Leu Gln Pro       Glu Asp                                                                                  50         - #         55         - #         60                      - -      Phe Ala Asp Tyr Phe Cys Phe Gly - # Gly Gly Thr Lys Leu Glu       Ile Lys                                                                              65             - #     70             - #     75             - #         80                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - -      Asp Ile Val Met Thr Gln Ser Pro - # Asp Ser Leu Ala Val Ser        Leu Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Arg Ala Thr Ile Asn Cys Trp - # Tyr Gln Gln Lys Pro Gly       Gln Pro                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Leu Leu Ile Tyr Gly Val - # Pro Asp Arg Phe Ser Gly        Ser Gly                                                                                      35     - #             40     - #             45                  - -      Ser Gly Thr Asp Phe Thr Leu Thr - # Ile Ser Ser Leu Gln Ala       Glu Asp                                                                                  50         - #         55         - #         60                      - -      Val Ala Val Tyr Tyr Cys Phe Gly - # Gln Gly Thr Lys Leu Glu       Ile Lys                                                                              65             - #     70             - #     75             - #         80                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - -      Asp Ile Val Met Thr Gln Ser Gln - # Lys Met Ser Thr Ser Val        Gly Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Arg Val Ser Ile Thr Cys Trp Tyr - # Gln Gln Lys Pro Gly Gln       Ser Pro                                                                                          20 - #                 25 - #                 30             - -      Lys Leu Leu Ile Tyr Gly Val Pro - # Asp Arg Phe Ser Gly Ser        Gly Ser                                                                                      35     - #             40     - #             45                  - -      Gly Thr Asp Phe Thr Leu Thr Ile - # Ser Asn Met Gln Ser Glu       Asp Leu                                                                                  50         - #         55         - #         60                      - -      Ala Asp Tyr Phe Cys Phe Gly Gly - # Gly Thr Lys Leu Glu Ile       Lys                                                                                  65             - #     70             - #     75                          - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - -      Asp Ile Val Met Thr Gln Ser Pro - # Asp Ser Leu Ala Val Ser       Leu Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Arg Ala Thr Ile Asn Cys Trp - # Tyr Gln Gln Lys Pro Gly       Gln Ser                                                                                          20 - #                 25 - #                 30             - -      Pro Lys Leu Leu Ile Tyr Gly Val - # Pro Asp Arg Phe Ser Gly        Ser Gly                                                                                      35     - #             40     - #             45                  - -      Ser Gly Thr Asp Phe Thr Leu Thr - # Ile Ser Ser Leu Gln Ala       Glu Asp                                                                                  50         - #         55         - #         60                      - -      Val Ala Asp Tyr Phe Cys Phe Gly - # Gly Gly Thr Lys Leu Glu       Ile Lys                                                                              65             - #     70             - #     75             - #         80                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - -      Gln Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Gly Ser Ile Ser       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Gln Pro Pro Gly Lys Gly Leu - # Glu Trp Ile Gly Arg Val        Thr Ile                                                                                      35     - #             40     - #             45                  - -      Ser Val Asp Thr Ser Lys Asn Gln - # Phe Ser Leu Lys Leu Ser       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Ala Ala Asp Thr Ala Val Tyr - # Tyr Cys Ala Arg Trp Gly       Gln Gly                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Thr Met Val Thr Val Ser Ser                                                           - #   85                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Ser Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Ser Val - # Thr Gly Asp Ser Ile Thr       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Lys Phe Pro Gly Asn Lys Leu - # Glu Tyr Met Gly Arg Ile        Ser Ile                                                                                      35     - #             40     - #             45                  - -      Thr Arg Asp Thr Ser Gln Asn Gln - # Phe Tyr Leu Gln Leu Asn       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Thr Glu Asp Thr Gly Thr Tyr - # Tyr Cys Ala Cys Trp Gly       Gln Gly                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Thr Thr Leu Thr Val Ser Ser                                                           - #   85                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Asp Ser Ile Thr       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Lys Pro Pro Gly Asn Lys Leu - # Glu Tyr Met Gly Arg Ile        Ser Ile                                                                                      35     - #             40     - #             45                  - -      Ser Arg Asp Thr Ser Lys Asn Gln - # Phe Ser Leu Lys Leu Ser       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Ala Ala Asp Thr Gly Val Tyr - # Tyr Cys Ala Cys Trp Gly       Gln Gly                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Thr Thr Leu Thr Val Ser Ser                                                           - #   85                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - -      Gln Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Gly Ser Ile Ser       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Gln Pro Ala Gly Lys Gly Leu - # Glu Trp Ile Gly Arg Val        Thr Ile                                                                                      35     - #             40     - #             45                  - -      Ser Val Asp Thr Ser Lys Asn Gln - # Phe Ser Leu Lys Leu Ser       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Ala Ala Asp Thr Ala Val Tyr - # Tyr Cys Ala Arg Trp Gly       Gln Gly                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Thr Met Val Thr Val Ser Ser                                                           - #   85                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - -      Gln Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Gly Ser Ile Ser       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Gln His Pro Gly Lys Gly Leu - # Glu Trp Ile Gly Arg Val        Thr Ile                                                                                      35     - #             40     - #             45                  - -      Ser Val Asp Thr Ser Lys Asn Gln - # Phe Ser Leu Lys Leu Ser       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Ala Ala Asp Thr Ala Val Tyr - # Tyr Cys Ala Arg Trp Gly       Gln Gly                                                                              65             - #     70             - #     75             - #         80                                                                            - -      Thr Thr Leu Thr Val Ser Ser                                                           - #   85                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - -      Glu Val Gln Leu Gln Glu Ser Gly - # Pro Gly Leu Val Lys Pro        Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Ser Leu Thr Cys Thr Val - # Ser Gly Asp Ser Ile Thr       Trp Ile                                                                                          20 - #                 25 - #                 30             - -      Arg Lys His Pro Gly Asn Lys Leu - # Glu Tyr Met Gly Arg Ile        Ser Ile                                                                                      35     - #             40     - #             45                  - -      Ser Arg Asp Thr Ser Lys Asn Gln - # Phe Ser Leu Lys Leu Ser       Ser Val                                                                                  50         - #         55         - #         60                      - -      Thr Ala Ala Asp Thr Gly Val Tyr - # Tyr Cys Ala Cys                      65             - #     70             - #     75                          - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 30                                                              (D) OTHER INFORMATION: - #/note= "Nucleotide 30 wherein N =                  (Ck)."                                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - TGCAGCATCC GTACGTTTGA TTCCAGCTTN         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #/note= "Nucleotide 20 wherein N = A                      or C."                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 21                                                              (D) OTHER INFORMATION: - #/note= "Nucleotide 21 wherein N = G                      or A."                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 27                                                              (D) OTHER INFORMATION: - #/note= "Nucleotide 27 wherein N = G                      or A."                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 32                                                              (D) OTHER INFORMATION: - #/note= "Nucleotide 32 wherein N = C                      gamma 1."                                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - GGGGGTGTCG TGCTAGCTGN NGAGACNGTG AN       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 411 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..411                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - AGATCTCTCA CC ATG GGC TTC AAG ATG GAG TCA CAG - # TTT CTG GCC TTT             48                                                                                     Met Gly Ph - #e Lys Met Glu Ser Gln Phe Leu Ala Phe                             1  - #             5     - #             10                    - - GTA TTC GCG TTT CTC TGG TTG TCT GGT GTT GA - #T GGA GAC ATT GTG ATG           96                                                                       Val Phe Ala Phe Leu Trp Leu Ser Gly Val As - #p Gly Asp Ile Val Met                    15         - #         20         - #         25                      - - ACC CAG TCT CCA TCT TTC CTC TCC GCC TCC GT - #A GGA GAC AGG GTC ACC          144                                                                       Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Va - #l Gly Asp Arg Val Thr                30             - #     35             - #     40                          - - ATC ACC TGC AAG GCC AGT CAG AAT GTG ATT AC - #T GCT GTA GCC TGG TAT          192                                                                       Ile Thr Cys Lys Ala Ser Gln Asn Val Ile Th - #r Ala Val Ala Trp Tyr            45                 - # 50                 - # 55                 - # 60       - - CAA CAG AAA CCA GGA AAG TCT CCT AAA TTG CT - #G ATT TAC TCG GCA TCC          240                                                                       Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Le - #u Ile Tyr Ser Ala Ser                            65 - #                 70 - #                 75              - - AAT CGG TAC ACT GGA GTC CCT GAT CGC TTC TC - #A GGC AGT GGG TCT GGG          288                                                                       Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Se - #r Gly Ser Gly Ser Gly                        80     - #             85     - #             90                  - - ACA GAT TTC ACT CTC ACC ATC AGC TCT CTC CA - #G CCA GAA GAC TTC GCA          336                                                                       Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gl - #n Pro Glu Asp Phe Ala                    95         - #        100         - #        105                      - - GAT TAT TTC TGC CAG CAA TAT AAC AGC TAT CC - #G TAC ACG TTC GGA GGG          384                                                                       Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pr - #o Tyr Thr Phe Gly Gly               110              - #   115              - #   120                          - - GGG ACC AAG CTG GAA ATC AAA CGT ACG    - #                  - #                411                                                                     Gly Thr Lys Leu Glu Ile Lys Arg Thr                                           125                 1 - #30                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 411 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..411                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - AGATCTCTCA CC ATG GGC TTC AAG ATG GAG TCA CAG - # TTT CTG GCC TTT             48                                                                                     Met Gly Ph - #e Lys Met Glu Ser Gln Phe Leu Ala Phe                               - #135                 - #140                 - #145         - - GTA TTC GCG TTT CTC TGG TTG TCT GGT GTT GA - #T GGA GAC ATT GTG ATG           96                                                                       Val Phe Ala Phe Leu Trp Leu Ser Gly Val As - #p Gly Asp Ile Val Met                           150  - #               155  - #               160              - - ACC CAG TCT CCA GAT TCT CTC GCC GTG TCC CT - #C GGA GAG AGG GCC ACC          144                                                                       Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Le - #u Gly Glu Arg Ala Thr                       165      - #           170      - #           175                  - - ATC AAC TGC AAG GCC AGT CAG AAT GTG ATT AC - #T GCT GTA GCC TGG TAT          192                                                                       Ile Asn Cys Lys Ala Ser Gln Asn Val Ile Th - #r Ala Val Ala Trp Tyr                   180          - #       185          - #       190                      - - CAA CAG AAA CCA GGA CAA TCT CCT AAA TTG CT - #G ATT TAC TCG GCA TCC          240                                                                       Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Le - #u Ile Tyr Ser Ala Ser               195              - #   200              - #   205                          - - AAT CGG TAC ACT GGA GTC CCT GAT CGC TTC TC - #A GGC AGT GGG TCT GGG          288                                                                       Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Se - #r Gly Ser Gly Ser Gly           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - ACA GAT TTC ACT CTC ACC ATC AGC TCT CTC CA - #G GCC GAA GAC GTG        GCA      336                                                                    Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gl - #n Ala Glu Asp Val Ala                          230  - #               235  - #               240              - - GAT TAT TTC TGC CAG CCA TAT AAC AGC TAT CC - #G TAC ACG TTC GGA GGG          384                                                                       Asp Tyr Phe Cys Gln Pro Tyr Asn Ser Tyr Pr - #o Tyr Thr Phe Gly Gly                       245      - #           250      - #           255                  - - GGG ACC AAG CTG GAA ATC AAA CGT ACG    - #                  - #                411                                                                     Gly Thr Lys Leu Glu Ile Lys Arg Thr                                                   260          - #       265                                             - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 426 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..426                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - GTCGAC ATG ATG GTG TTA AGT CTT CTG TAC CTG - #TTG ACA GCC CTT CCG            48                                                                               Met Met Val Leu Ser Leu Le - #u Tyr Leu Leu Thr Ala Leu Pro                     1         - #      5            - #      10                           - - GGT TTC CTG TCA GAG GTG CAG CTT CAG GAG TC - #A GGA CCT GGC CTC GTG           96                                                                       Gly Phe Leu Ser Glu Val Gln Leu Gln Glu Se - #r Gly Pro Gly Leu Val            15                 - # 20                 - # 25                 - # 30       - - AAA CCT TCT GAG ACT CTG TCC CTC ACC TGT AC - #C GTC TCT GGC GAC TCC          144                                                                       Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Th - #r Val Ser Gly Asp Ser                            35 - #                 40 - #                 45              - - ATC ACT AAT GGT TTC TGG ATC TGG ATC CGG AA - #A CCA CCA GGG AAT AAA          192                                                                       Ile Thr Asn Gly Phe Trp Ile Trp Ile Arg Ly - #s Pro Pro Gly Asn Lys                        50     - #             55     - #             60                  - - CTT GAG TAC ATG GGC TAC ATA AGT TAC AGT GG - #T AGC ACT TAC TAC AAT          240                                                                       Leu Glu Tyr Met Gly Tyr Ile Ser Tyr Ser Gl - #y Ser Thr Tyr Tyr Asn                    65         - #         70         - #         75                      - - CCA TCT CTC AAG AGT CGA ATC TCC ATC TCT CG - #C GAC ACA TCC AAG AAC          288                                                                       Pro Ser Leu Lys Ser Arg Ile Ser Ile Ser Ar - #g Asp Thr Ser Lys Asn                80             - #     85             - #     90                          - - CAG TTC TCT CTA AAG TTG TCT TCT GTG ACT GC - #C GCC GAC ACA GGC GTG          336                                                                       Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Al - #a Ala Asp Thr Gly Val            95                 - #100                 - #105                 - #110       - - TAT TAC TGT GCC TGC CGC AGT TAC GGG AGG AC - #C CCG TAC TAC TTT GAC          384                                                                       Tyr Tyr Cys Ala Cys Arg Ser Tyr Gly Arg Th - #r Pro Tyr Tyr Phe Asp                           115  - #               120  - #               125              - - TTC TGG GGC CAA GGC ACC ACT CTC ACC GTC TC - #C TCA GCT AGC                 - # 426                                                                    Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Se - #r Ser Ala Ser                               130      - #           135      - #           140                  - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 411 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..411                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - AGATCTCTCA CC ATG GGC TTC AAG ATG GAG TCA CAG - # TTT CTG GCC TTT             48                                                                                     Met Gly Ph - #e Lys Met Glu Ser Gln Phe Leu Ala Phe                               - #135                 - #140                 - #145         - - GTA TTC GCG TTT CTC TGG TTG TCT GGT GTT GA - #T GGA GAC ATT GTG ATG           96                                                                       Val Phe Ala Phe Leu Trp Leu Ser Gly Val As - #p Gly Asp Ile Val Met                           150  - #               155  - #               160              - - ACC CAG TCT CAA AAA TTC ATG TCC ACA TCC GT - #A GGA GAC AGG GTC AGC          144                                                                       Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Va - #l Gly Asp Arg Val Ser                       165      - #           170      - #           175                  - - ATC ACC TGC AAG GCC AGT CAG AAT GTG ATT AC - #T GCT GTA GCC TGG TAT          192                                                                       Ile Thr Cys Lys Ala Ser Gln Asn Val Ile Th - #r Ala Val Ala Trp Tyr                   180          - #       185          - #       190                      - - CAA CAG AAA CCA GGA CAA TCT CCT AAA TTG CT - #G ATT TAC TCG GCA TCC          240                                                                       Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Le - #u Ile Tyr Ser Ala Ser               195              - #   200              - #   205                          - - AAT CGG TAC ACT GGA GTC CCT GAT CGC TTC TC - #A GGC AGT GGG TCT GGG          288                                                                       Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Se - #r Gly Ser Gly Ser Gly           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - ACA GAT TTC ACT CTC ACC ATC AGC AAT ATG CA - #G TCT GAA GAC CTG        GCA      336                                                                    Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gl - #n Ser Glu Asp Leu Ala                          230  - #               235  - #               240              - - GAT TAT TTC TGC CAG CAA TAT AAC AGC TAT CC - #G TAC ACG TTC GGA GGG          384                                                                       Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pr - #o Tyr Thr Phe Gly Gly                       245      - #           250      - #           255                  - - GGG ACC AAG CTG GAA ATC AAA CGT ACG    - #                  - #                411                                                                     Gly Thr Lys Leu Glu Ile Lys Arg Thr                                                   260          - #       265                                             - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 426 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..426                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - GTCGAC ATG ATG GTG TTA AGT CTT CTG TAC CTG - #TTG ACA GCC CTT CCG            48                                                                               Met Met Val Leu Ser Leu Le - #u Tyr Leu Leu Thr Ala Leu Pro                       135       - #          140       - #          145                   - - GGT TTC CTG TCA GAG GTG CAG CTT CAG GAG TC - #A GGA CCT AGC CTC GTG           96                                                                       Gly Phe Leu Ser Glu Val Gln Leu Gln Glu Se - #r Gly Pro Ser Leu Val                   150          - #       155          - #       160                      - - AAA CCT TCT CAG ACT CTG TCC CTC ACC TGT TC - #T GTC ACT GGC GAC TCC          144                                                                       Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Se - #r Val Thr Gly Asp Ser               165              - #   170              - #   175                          - - ATC ACT AAT GGT TTC TGG ATC TGG ATC CGG AA - #A TTC CCA GGG AAT AAA          192                                                                       Ile Thr Asn Gly Phe Trp Ile Trp Ile Arg Ly - #s Phe Pro Gly Asn Lys           180                 1 - #85                 1 - #90                 1 -      #95                                                                              - - CTT GAG TAC ATG GGC TAC ATA AGT TAC AGT GG - #T AGC ACT TAC TAC        AAT      240                                                                    Leu Glu Tyr Met Gly Tyr Ile Ser Tyr Ser Gl - #y Ser Thr Tyr Tyr Asn                          200  - #               205  - #               210              - - CCA TCT CTC AAG AGT CGA ATC TCC ATC ACT CG - #C GAC ACA TCC CAG AAC          288                                                                       Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Ar - #g Asp Thr Ser Gln Asn                       215      - #           220      - #           225                  - - CAG TTC TAC CTA CAA TTG AAT TCT GTG ACT AC - #T GAG GAC ACA GGC ACA          336                                                                       Gln Phe Tyr Leu Gln Leu Asn Ser Val Thr Th - #r Glu Asp Thr Gly Thr                   230          - #       235          - #       240                      - - TAT TAC TGT GCC TGC CGC AGT TAC GGG AGG AC - #C CCG TAC TAC TTT GAC          384                                                                       Tyr Tyr Cys Ala Cys Arg Ser Tyr Gly Arg Th - #r Pro Tyr Tyr Phe Asp               245              - #   250              - #   255                          - - TTC TGG GGC CAA GGC ACC ACT CTC ACC GTC TC - #C TCA GCT AGC                 - # 426                                                                    Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Se - #r Ser Ala Ser                   260                 2 - #65                 2 - #70                          __________________________________________________________________________

What is claimed is:
 1. A humanized antibody or antigen-binding fragmentthereof that specifically binds the CD40 ligand wherein said humanizedantibody or fragment thereof contains a variable light sequence whichcomprises the amino acid sequence encoded by the nucleic acid sequencehaving SEQ ID No. 24 or SEQ ID No. 25 and the said humanized antibody orfragment thereof contains a variable heavy sequence which comprises theamino acid sequence encoded by the nucleic acid sequence having SEQ IDNo.
 26. 2. The humanized antibody or antigas-binding fragment of claim1, wherein the variable light sequence has the amino acid sequenceencoded by the nucleic acid sequence having SEQ ID No.
 24. 3. Thehumanized antibody or antigas-binding fragment of claim 1, wherein thevariable ligand sequence has the amino acid sequence encoded by thenucleic acid sequence having SEQ ID No.
 25. 4. The humanized antibody ofclaim 1, which comprises human heavy and light constant domains.
 5. Thehumanized antibody of claim 4, wherein the human suitable heavy constantregion is selected from the group consisting of human gamma 1, humangamma 2, human gamma 3, human gamma
 4. 6. The humanized antibody ofclaim 4, wherein the human variable heavy constant region is gamma 4 ora mutated gamma 4 which contains either or both of the followingmodifications:(i) change of a leucine to a glutamic acid at position 236(according to the Kabat numbering system); and (ii) change of a serineto a proline at position 229 (according to the Kabat numbering system).7. The humanized antibody of claim 6, which comprises a mutated gamma 4containing both of said modifications.
 8. The humanized antibody ofclaim 4, wherein the human light constant region is the Kappa or lambdaconstant region.
 9. The humanized antibody according to claim 4 whichcomprises the human Kappa light chain constant region and human gamma 4heavy chain constant region or a mutated human gamma 4 heavy chainconstant region, wherein the mutated gamma 4 heavy chain compriseseither or both of the following modifications:(i) the change of aleucine to glutamic acid at position 236 according to the Kabatnumbering system; and (ii) the change of a serine to proline at position229 according to the Kabat numbering system.
 10. A humanized antibodyaccording to claim 1, which contains one or more of the followingmodifications:(i) substitution of the amino acid residue at one or moreof positions 34, 43, 44 and 68 of the variable heavy sequence (accordingto Kabat numbering scheme) with a different amino acid; (ii)substitution of residue 85 of the variable light chain (according to theKabat numbering scheme) with a different amino acid.
 11. The humanizedantibody according to the claim 1, which further comprises one or moreconservative amino acid substitutions which do not adversely affect CD40ligand binding.
 12. A pharmaceutical composition containing atherapeutically effective amount of a humanized antibody or antigenbinding domain according to claim 1, and a pharmaceutically acceptablecarrier.